CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. Provisional Application Serial No. 63/171,909, filed April 7, 2021. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named 07039-2020W01 Sequence. The ASCII text file, created on April 6, 2022, is 7 kilobytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This document relates to methods and materials for treating atherosclerosis. For example, this document provides methods and materials for using IGF-1 signaling pathway modulators to reverse atherogenic plaque instability.

BACKGROUND

Atherosclerotic lesions (plaques) can cause myocardial infarction or stroke, and as such, are a leading cause of death in the United States and many other developed countries. Systemic hypercholesteremia (dyslipidemia) results in the accumulation of oxidized-low density lipoprotein (LDL) in the subendothelial space, between the endothelium and vascular smooth muscle cell rich media of arterial blood vessels.

Despite tremendous success in treating atherosclerotic disease with lipid lowering drugs that slow the formation of end-stage lesions (also referred to as unstable or vulnerable plaques) that are at risk of rupture, platelet mediated arterial occlusion, and end-organ failure, a considerable proportion of patients die from disease progression (Bittencourt and Cerci, BMC Med 13:260, 2015; and Reith and Armitage, Atherosclerosis 245:161- 170, 2016).

SUMMARY

This document provides methods and materials for treating atherosclerosis. For example, this document provides methods and materials for using IGF-1 signaling pathway modulators to reverse atherogenic plaque instability. As described herein, atherosclerotic lesions, including end-stage lesions with highly degenerated, fragile fibrous cap structures, can be re-stabilized by administering one or more IGF-1 signaling pathway modulators to a mammal (e.g., a human). For example, one or more IGF-1 receptor activators (e.g., an IGF-1 or IGF-2 polypeptide) and/or one or more IGFBP-3 inhibitors (e.g., an anti-IGFBP-3 antibody) can be administered to a mammal to reverse cap-thinning and/or restore vascular smooth muscle cell (VSMC) migration and activity within the mammal, thereby stabilizing atherosclerotic lesions within the mammal.

In some cases, described herein are methods for reversing atherogenic plaque instability in a human in need thereof, where the methods include, or consist essentially of, administering to the human an IGF-1 signaling pathway modulator at an amount effective to increase a thickness of a fibrous cap of a thin-cap fibroatheroma, thereby reversing atherogenic plaque instability in the human. In some cases, the IGF-1 signaling pathway modulator is an IGF-1 receptor activator. In some cases, the IGF-1 receptor activator is an IGF-1 polypeptide or an IGF-2 polypeptide. In some cases, the IGF-1 receptor activator is a recombinant IGF-1 (rIGF-1) or recombinant LR3 IGF-1 polypeptide. In some cases, the IGF-1 signaling pathway modulator is an IGFBP-3 inhibitor. In some cases, the IGFBP-3 inhibitor decreases binding of IGFBP-3 to IGF-1. In some cases, the IGFBP-3 inhibitor is an IGFBP-3 antibody. In some cases, the IGFBP-3 inhibitor is an antisense oligonucleotide or an expression construct that encodes an IGFBP-3 neutralizing antibody.

In some cases, described herein are methods for reversing atherogenic plaque instability in a human in need thereof, where the methods include, or consist essentially of, administering to the human an IGF-1 signaling pathway modulator at an amount effective to increase a thickness of a fibrous cap of a thin-cap fibroatheroma, wherein the thin-cap fibroatheroma has a fibrous cap thickness of less than about 150 pm, thereby reversing atherogenic plaque instability in the human. In some cases, the thin-cap fibroatheroma has a fibrous cap thickness of less than about 15 pm. In some cases, the methods described herein further include measuring a thickness of the fibrous cap of the thin-cap fibroatheroma prior to administering the IGF-1 signaling pathway modulator to the human. In some cases, the measuring includes intravascular optical coherence tomography (OCT). In some cases, the IGF-1 signaling pathway modulator is an expression construct that encodes an IGF-1 polypeptide. In some cases, the administering includes systemically administering the IGF-1 signaling pathway modulator to the human. In some cases, the administering includes locally administering the IGF-1 signaling pathway modulator to the human.

In some cases, described herein are methods for reversing atherogenic plaque instability in a human in need thereof, where the methods include, or consist essentially of, administering to the human an IGF-1 signaling pathway modulator at an amount effective to increase a thickness of a fibrous cap of a thin-cap fibroatheroma, thereby reversing atherogenic plaque instability in the human, wherein reversing atherogenic plaque instability in the human includes reducing occurrence of: rupture of the atherogenic plaque, myocardial infarction in the human, ischemic stroke in the human, angina in the human, peripheral vascular disease in the human, or a combination thereof. In some cases, the methods described herein include administering the IGF-1 signaling pathway modulator in combination with a lipid-lowering agent. In some cases, the lipid lowering agent is a statin.

In some cases, described herein are methods for reversing atherogenic plaque instability in a human in need thereof, where the methods include, or consist essentially of, administering to the human an IGF-1 signaling pathway modulator at an amount effective to increase a thickness of a fibrous cap of a thin-cap fibroatheroma, thereby reversing atherogenic plaque instability in the human, wherein reversing atherogenic plaque instability in the human includes increasing an amount of vascular smooth muscle cells (VSMCs) within the fibrous cap, increasing an elastin content within the fibrous cap, increasing an elastin content within the fibrous cap, increasing a thickness of the fibrous cap, or a combination thereof. In some cases, the IGF-1 signaling pathway modulator decreases an IFG-1 receptor nuclear translocation. In some cases, the IGF-1 signaling pathway modulator upregulates a gene involved in VSMC pro migratory phenotype switching. In some cases, the gene involved in VSMC promigratory phenotype switching is selected from the group consisting of Vwc2, EdnRA, LepR, Bmp3, Prdm6, Tshz3, Igfbp6, 1133, Aldhla2, and Lyvel. In some cases, the IGF-1 signaling pathway modulator downregulates a gene involved in VSMC promigratory phenotype switching. In some cases, the gene involved in VSMC promigratory phenotype switching is selected from the group consisting of Sp7, Col2al, CollOal, Ibsp, Clec3a, Tgml, Chad, Ccl5, Prfl, Ltb, Hfe, and SerpinBlO.

In some cases, disclosed herein are methods for reversing atherogenic plaque instability in a human undergoing statin therapy in need thereof, where the methods include, or consist essentially of, administering to the human undergoing statin therapy an IGF-1 signaling pathway modulator at an amount effective to increase a thickness of a fibrous cap of a thin-cap fibroatheroma, thereby reversing atherogenic plaque instability in the human. In some cases, the statin therapy includes a statin selected from the group consisting of lovastatin, pravastatin, simvastatin, atorvastatin, cerivastatin, Fluvastatin, pravastatin, pitavastatin, and rosuvastatin.

In some cases, described herein are methods for identifying an agent that increases atherogenic plaque stability, where the methods include, or consist essentially of, measuring a thickness of a fibrous cap of an atherogenic plaque; contacting the atherogenic plaque with a candidate agent; and determining a change in the thickness of the fibrous cap, wherein when the thickness of the fibrous cap increases, the candidate agent is an agent that increases atherogenic plaque stability. In some cases, the candidate agent is an IGF-1 receptor activator. In some cases, the IGF-1 receptor activator is an expression construct that encodes an IGF-1. In some cases, the candidate agent is an IGFBP-3 inhibitor. In some cases, the IGFBP-3 inhibitor is an antisense oligonucleotide or an expression construct that encodes an IGFBP-3 neutralizing antibody. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1G show lesional IGFBP-3’s role in promigratory switching of medial

VSMCs. FIG. 1A includes a representative image (left) of IGFBP-3 ⁺ /SA b-GaF lesional cells (arrowheads), and a graph (right) plotting IGFBP-3 ⁺ cell frequency among SA b-

Gal ⁺ and SA b-Gal- cells from indicated groups. FIG. IB includes representative images

(left) of VSMCs outgrowing from aortic rings (AR) of wildtype C57BL/6 mice treated with the indicated conditioned media (CM), and a graph (right) plotting the number of outgrowing VSMCs in the indicated treatment groups. FIG. 1C includes representative images (left) of human aortic VSMCs emigrating into scratch wound space with the indicated conditioned media. Dashed lines indicate cell monolayer/scratch wound boundary. FIG. 1C also includes a graph (right) plotting emigrating VSMCs in the indicated experimental groups. LR3 IGF-1, long R3 mutant stabilized recombinant IGF-

1. FIG. ID is a schematic showing a 33-day HFD experiment in dlr mice to assess the effects of LR3 IGF-1. FIG. IE includes images (left) showing representative SMA immunostaining in lesions of 33-day HFD-fed Ldlr ^! mice treated with LR3 IGF-1 or corresponding Veh, and a graph (right) plotting quantification of neointimal Sma ⁺ cells in these mice. FIG. IF is a graph plotting quantification of cells crossing the first elastic fiber in indicated groups. FIG. 1G includes representative images (left) of SA b- Gal stained d!r aortic arches from mice fed HFD for 33 d with concurrent administration of either LR3 IGF1 or vehicle control, as well as graphs plotting quantification of the total plaque burden in the aortic arch (middle) and the percentage of aortic arch plaque with SA b-Gal positivity (right) in the indicated treatment groups. Statistics for the data in FIGS. 1A-1C were performed by ordinary one-way ANOVA with Holm-Sidak multiple comparison correction. All other panels were analyzed by unpaired, two-tailed /-test with Welch’s correction. Error bars represent s.e.m. *,p <0.05; **,p <0.01; ***, > <0.001. “n” refers to individual mice in FIGS. 1A-1B, IE, and 1G; and CM from independent cells lines in FIG. 1C. The scale bar in FIG. 1 A is 10 pm; FIG. IB, 500 pm; FIG. 1C, 200 pm; FIG. IE (main and inset), 20 pm; and FIG. 1G, 1 mm.

FIGS. 2A-2C show that senescent cell-derived IGFBP-3 inhibited IGF1- mediated promigratory phenotype switching of VSMCs. FIG. 2 A is a graph showing expression of Igfbp3 in the indicated MEFs measured by RT-qPCR analysis. FIG. 2B includes representative images showing immunofluorescent staining of VIM and SMA in human aortic VSMC with the indicated treatments. CM, conditioned media. FIG. 2C includes a pair of graphs plotting the average fluorescent signal intensity of SMA (left) and VIM (right) per cell in human aortic VSMC receiving the indicated treatments “n” refers in all panels to distinct lines of conditioned media. Analysis in FIG. 2A was performed with unpaired, two- tailed /-test; analyses in FIG. 2B and 2C were performed by ordinary one-way ANOVA with Holm-Sidak multiple comparison correction. Error bars represent s.e.m. *,p <0.05; **,p <0.01; ***, > <0.001. The scale bar in FIG. 2B is 100 pm.

FIGS 3A-3E show that IGFBP-3 neutralization promoted promigratory switching of VSMC in mouse and human explant atheromas. FIG. 3A is a schematic showing an in vitro IGFBP-3 neutralization experiment in aortic arch plaque from Ldlr ^! mice. FIG. 3B includes representative images (left) of explanted lesions from Ldlr ^! mice cultured in the presence of the indicated antibodies and costained for Vim and Sma. Neointima flanking the media (lesion) and IFS1-3 are shown. FIG. 3B also includes a graph (right) plotting the percentage Sma ⁺ /Vim and Sma /Vim ⁺ cells in IFS1 of the indicated explants. Vim ⁺ /Sma ⁺ frequency is not shown, as it was unchanged with treatment. FIG. 3C is a schematic of an in vitro IGFBP-3 neutralization experiment in aortic and femoral plaque from human endarterectomy patients. FIG. 3D includes representative images (left) of the indicated explanted human plaque cultured in the presence of the indicated antibodies and costained for Vim and Sma, and a graph (right) plotting colocalization of Vim and Sma signal in human plaque treated in this fashion. FIG. 3E includes graphs plotting colocalization of Vim and Sma signal in human plaque treated in this fashion. Statistics for FIGS. 3B and 3D were performed by unpaired, two-tailed /-test with Welch’ s correction. Statistics for FIG. 3E were performed using ordinary one-way ANOVA with Holm-Sidak multiple comparison correction (both Ab224530 and PA529711 versus IgG); IgG data is duplicated for ease of display across the two panels in FIG. 3E. Error bars represent s.e.m. “n” represents explanted aortic plaque-bearing rings in FIG. 3B, and endarterectomy patients in FIGS. 3D and 3E. *,p < 0.05; **,p <0.01. Scale bars in FIGS. 3B, 3D, and 3E are 50 pm.

FIG. 4 illustrates a model for how SNCs can suppress the cap repair functions of VSMCs in advanced atherosclerotic lesions. (Top) SNCs inhibit lesional IGF signaling by elevating IGFBP3 levels, thereby suppressing promigratory phenotype switching of contractile VSMCs in the media and their recruitment to the fibrous cap, as well as ECM deposition by VSMCs in the cap, resulting in fibrous cap erosion. (Bottom) Reducing lesional IGFBP-3 levels stimulates the IGF-mediated promigratory phenotype switching of medial VSMCs and their recruitment to the fibrous cap, as well as ECM deposition by VSMCs in the cap, restoring fibrous cap thickness and plaque stability.

FIG. 5 depicts a graph plotting colocalization of Vim and Sma signal in human plaques cultured in the presence of the indicated antibodies and sorted by subject receiving statin therapy (circles) or subjects not receiving statin therapy (triangle). DETAILED DESCRIPTION

Atherosclerotic lesions have a specialized plaque-covering fibrous cap that is established as part of the vessel injury response to atherogenesis. Fibrous cap thinning is a degenerative process that can destabilize plaques and promote myocardial infarction and stroke. Reparative VSMCs are a major cell type within the fibrous cap. As described herein, SNCs can antagonize tissue repair processes to drive aging-related disease. In the work described in the Examples herein, pharmacological and transgenic approaches were used in highly advanced lesions of the Ldlr ^f mouse model of atherosclerosis. These studies demonstrated that SNCs prompt cap thinning by targeting reparative VSMCs, suggesting that in addition to stimulating tissue repair (e.g., in wound healing), SNCs also can act to inhibit repair mechanisms. In addition, the Examples herein demonstrated that the senescence associated secretory phenotype (SASP) attenuates dedifferentiation of medial VSMCs in vitro by altering signaling through the IGF-IGFBP axis, as exemplified by the observation that various reagents that normalize this axis restored VSMC phenotype switching. Thus, the work described herein demonstrates a new, therapeutically-targetable biology in atherosclerosis.

In some cases, this document provides methods and materials for treating atherosclerosis. For example, this document provides methods and materials for using one or more IGF-1 signaling pathway modulators to treat a mammal having atherosclerosis (e.g., to reverse atherogenic plaque instability, to reduce the risk or rate of atherosclerosis progression, to reduce the risk of atherosclerosis reoccurrence, to increase the thickness of a fibrous cap over an atherosclerotic plaque, and/or to promote repair of a fibrous cap over an atherosclerotic lesion). In some cases, one or more IGF-1 signaling pathway modulators can be administered to a mammal (e.g., a human) having or having had atherosclerosis, to reduce the risk of further atherogenesis.

Any appropriate mammal having atherosclerosis, at risk of developing atherosclerosis, or having had atherosclerosis, can be treated as described herein. Examples of mammals that can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, rabbits, and rats. In some cases, a human having atherosclerosis, at risk of developing atherosclerosis, or having had atherosclerosis can be treated as described herein.

When treating a mammal (e.g., a human) having atherosclerosis, at risk of developing atherosclerosis, or having had atherosclerosis as described herein, the atherosclerotic lesion(s) can be at any stage of progression. In some cases, an atherosclerotic lesion treated as described herein can be an established, but not end-stage lesion. In some cases, an atherosclerotic lesion treated as described herein can be an end- stage lesion. In some cases, an atherosclerotic lesion as described herein is predicted to rupture. In some cases, an atherosclerotic lesion predicted to rupture as described herein has a fibrous cap with a thickness, prior to treatment, of 150 pm. In some cases, an atherosclerotic lesion treated as described herein can have fibrous cap with a thickness, prior to treatment, that is less than about 200 pm (e.g., less than about 150 pm, less than about 100 pm, less than about 80 pm, less than about 65 pm, less than about 50 pm, less than about 15 pm, or less than about 10 pm). Atherosclerotic lesions having a thickness less than about 200 pm can be referred to as “thin-cap fibro atheromas.” Any suitable method can be used to determine the thickness of a fibrous cap. For example, intravascular optical coherence tomography (OCT) can be used to measure the thickness of a fibrous cap over an atherosclerotic lesion, before and/or after treatment as described herein.

In some cases, the methods described herein can include identifying a mammal (e.g., a human) as having atherosclerosis. Any appropriate method can be used to identify a mammal as having atherosclerosis. For example, techniques such as blood tests for cholesterol and sugar levels, ultrasound, ankle-brachial index, stress test, angiogram, echocardiogram (ECG), computerized tomography (CT) scan, and magnetic resonance angiogram (MRA) can be used to identify mammals (e.g., humans) having atherosclerosis.

In some cases, a mammal having atherosclerosis or at risk of developing atherosclerosis can be administered or instructed to self-administer one or more (e.g., one, two, three, four, five, six, or more) IGF-1 signaling pathway modulators to treat the atherosclerosis (e.g., to reverse plaque instability, to reduce the risk or rate of atherosclerosis progression, to reduce the risk of atherosclerosis reoccurrence, to increase the thickness of a fibrous cap over an atherosclerotic plaque, and/or to promote repair of a fibrous cap over an atherosclerotic lesion).

Any appropriate IGF-1 signaling pathway modulator can be administered as described herein to treat atherosclerosis (e.g., to reverse atherogenic plaque instability, to reduce the risk or rate of atherosclerosis progression, to reduce the risk of atherosclerosis reoccurrence, to increase the thickness of a fibrous cap over an atherosclerotic plaque, and/or to promote repair of a fibrous cap over an atherosclerotic lesion). Examples of IGF-1 signaling pathway modulators that can used as described herein include, without limitation, IGF-1 receptor activators (e.g., IGF-1 polypeptides, including recombinant IGF-1 (rIGF-1) or recombinant LR3 IGF-1 polypeptides, and IGF-2 polypeptides), agents that decrease IFG-1 receptor nuclear translocation (e.g., dansylcadaverine), IGFBP-3 inhibitors (e.g., agents that can reduce binding of IGFBP-3 to IGF-1), such as anti- IGFBP-3 antibodies (e.g., IGFBP-3 neutralizing antibodies), enzymes that degrade IGFBP-3, and IGFBP-3 antisense oligonucleotides (e.g., oligonucleotides complementary to the 20 nucleotides that encode the N terminus of IGFBP-3, including 5 '-CAT GAC GCC TGC AAC CGG GG-3 '.

In some cases, an IGF-1 or IGF-2 polypeptide, or a nucleic acid encoding an IGF- 1 polypeptide (e.g., an LR3 IGF-1 polypeptide) or an IGF-2 polypeptide can be administered to a mammal. For example, an expression construct containing a nucleic acid sequence encoding an IGF-1 polypeptide, an LR3 IGF-1 polypeptide, or an IGF-2 polypeptide can be administered to a mammal having atherosclerosis to treat atherosclerosis as described herein. Representative amino acid sequences for IGF-1 (Uniprot P05019), IGF-2 (Uniprot P01344), and LR3 IGF-1 polypeptides are set forth in Table 1 below.

Table 1: Representative amino acid sequences for IGF-1, IGF-2, and LR3 IGF-1

In some cases, an anti-IGFBP-3 antibody (e.g., an IGFBP-3 neutralizing antibody), or an expression construct encoding an anti-IGFBP-3 antibody, can be administered to a mammal having atherosclerosis to treat atherosclerosis as described herein. For example, an anti-IGFBP-3 antibody can be an antibody that blocks phosphorylation to one or more residues in the central domain of IGFBP-3 (see, e.g., Gupta, J Cell Commun Signal 9(2): 111-123, 2015). In some cases, the anti-IGFBP-3 antibody is an antibody obtained via immunization with a synthetic phosphopeptide corresponding to amino acid residues surrounding S183 of human IGFBP3 (e.g., AB76001 available from ABCAM). In some cases, the anti-IGFBP-3 antibody is an antibody obtained via immunization with a recombinant fragment (e.g., AB224530 available from ABCAM). In some cases, the anti-IGFBP-3 antibody is an antibody obtained via immunization with a recombinant fragment corresponding to a region within amino acids 84 and 291 of human IGFBP3 (e.g., PA5-29711 available from THERMOFISHER SCIENTIFIC).

Any appropriate systemic or local method can be used to administer one or more

IGF-1 signaling pathway modulators (e.g., an IGF-1 receptor activator or an IGFBP-3 inhibitor) to a mammal (e.g., a mammal such as a human having atherosclerosis). In some cases, one or more IGF-1 signaling pathway modulators can be systemically administered to a mammal by an oral or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intradermal) route. In some cases, one or more IGF-1 signaling modulators can be administered locally to the vicinity of an atherogenic plaque. Methods for local administration include, without limitation, use of a stent or use of a catheter.

In some cases, when treating a mammal (e.g., a human) having atherosclerosis as described herein, the treatment can be effective to reverse instability of an atherosclerotic lesion in the mammal, effective to reduce the rate or risk of atherosclerosis progression, effective to reduce the risk of atherosclerosis reoccurrence, effective to reduce the number of atherosclerotic plaques in the mammal, effective to increase the thickness of a fibrous cap over an atherosclerotic plaque in the mammal, and/or effective to promote repair of a fibrous cap over an atherosclerotic lesion in the mammal. For example, the thickness of a fibrous cap over an atherosclerotic lesion can be increased by at least about 50% (e.g., at least about 60%, at least about 75%, at least about 90%, at least about 100%, at least about 200%, or at least about 300%) following administration of one or more IGF-1 signaling pathway modulators (e.g., administration of an anti-IGFBP-3 antibody) as described herein. In some cases, administration of one or more IGF-1 signaling pathway modulators eliminates regions of the fibrous cap that are prone to rupture (e.g., regions of the fibrous cap with a thickness of less than about 150 pm). In some cases, the size of one or more atherosclerotic lesions present within a mammal can be reduced using the materials and methods described herein. In some cases, the materials and methods described herein can be used to reduce the size (e.g., width at its largest point) of one or more atherosclerotic lesions present within a mammal by, for example, at least 10 percent (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 95 percent). In some cases, the materials and methods described herein can be used such that the size (e.g., width at its largest point) of one or more atherosclerotic lesions present within a mammal does not increase. In some cases, the number of atherosclerotic lesions present within a mammal can be reduced using the materials and methods described herein. For example, one or more IGF-1 signaling pathway modulators (e.g., an anti-IGFBP-3 antibody) can be administered to reduce the number of atherosclerotic lesions present within a mammal by, for example, at least 25 percent (e.g., at least 50 percent, at least 75 percent, or 100 percent).

In some cases, one or more IGF-1 signaling pathway modulators can be used to upregulate expression of a gene involved in VSMC promigratory phenotype switching. For example, one or more IGF-1 signaling pathway modulators can be administered to a mammal having atherosclerosis to increase expression (e.g., at the transcriptional or translational level) of one or more genes selected from Vwc2, EdnRA, LepR , Bmp3, Prdm6 , Tshz3 , Igfbp6 , 1133, Aldhla2, and Lyvel. In some cases, one or more IGF-1 signaling pathway modulators can be used to downregulate expression of a gene involved in VSMC promigratory phenotype switching. For example, one or more IGF-1 signaling pathway modulators can be administered to a mammal having atherosclerosis to decrease expression (e.g., at the transcriptional or translational level) of one or more genes selected from Sp7, Col2al, CollOal, Ibsp, Clec3a, Tgml, Chad, Ccl5, Prfl , Ltb, Hfe, and SerpinBlO.

In some cases, treating a mammal as described herein can be effective to reduce the likelihood or occurrence of one or more of the following: rupture of an atherogenic plaque, myocardial infarction, ischemic stroke, angina, and peripheral vascular disease.

In some cases, the methods provided herein can be effective to increase the amount of VSMCs within a fibrous cap and/or to increase the elastin content within a fibrous cap. In some cases, the amount of VSMCs within a fibrous cap and/or the elastin content within a fibrous cap is assessed by optical coherence tomography (OCT). In some cases, when treating a mammal (e.g., a human) having atherosclerosis as described herein, the treatment can be effective to improve survival of the mammal. For example, disease-free survival (e.g., relapse-free survival) can be improved using the materials and methods described herein. In some cases, progression-free survival can be improved using the materials and methods described herein. In some cases, the materials and methods described herein can be used to improve the survival of a mammal having atherosclerosis by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

In some cases, a mammal having, at risk of developing, or having had atherosclerosis can be treated by administering one or more IGF- 1 signaling pathway modulators as described herein and by administering one or more (e.g., one, two, three, four, five, or more) lipid-lowering agents and/or therapies. In some cases, the one or more lipid-lowering agents and/or therapies can be administered together with the one or more IGF-1 signaling pathway modulators. In some cases, the one or more (e.g., one, two, three, four, five, or more) lipid-lowering agents and/or therapies can be administered independent of the one or more IGF-1 signaling pathway modulators. When the one or more lipid lowering agents and/or therapies are administered independent of the one or more IGF-1 signaling pathway modulators, the one or more IGF-1 signaling pathway modulators can be administered first, and the one or more lipid-lowering agents and/or therapies administered second, or vice versa.

Any appropriate lipid-lowering agent and/or therapy can be used in combination with one or more IGF-1 signaling pathway modulators (e.g., an IGF-1 receptor activator or an IGFBP-3 inhibitor, such as an IGFBP-3 neutralizing antibody) as described herein. Examples of molecules that can lower lipid levels within a mammal include, without limitation, statins (e.g., lovastatin, pravastatin, simvastatin, atorvastatin, cerivastatin, Fluvastatin, pravastatin, pitavastatin, or rosuvastatin), fibrates (e.g., clofibrate, gemfibrozil, or fenofibrate), niacin, lecithin, bile acid sequestrants (e.g., cholestyramine, colesevelam, or colestipol), inhibitors of dietary cholesterol absorption (e.g., ezetimibe or Sch-48461), triglyceride transfer protein inhibitors, phytosterols, omega-3 supplements, or PCSK9 inhibitors (e.g., alirocumab or evolocumab). In some embodiments, the methods disclosed herein can include administering to a human receiving a lipid lowering agent an IGF-1 signaling pathway modulator at an effective amount to increase a thickness of a fibrous cap of a thin-cap fibroatheroma, thereby reversing atherogenic plaque instability. In some embodiments, the methods disclosed herein can include administering to a human undergoing statin therapy an IGF-1 signaling pathway modulator at an effective amount to increase a thickness of a fibrous cap of a thin-cap fibroatheroma, thereby reversing atherogenic plaque instability. In some embodiments, the human undergoing statin therapy is receiving a statin selected from the group consisting of lovastatin, pravastatin, simvastatin, atorvastatin, cerivastatin, Fluvastatin, pravastatin, pitavastatin, and rosuvastatin. In some cases, one or more IGF-1 signaling pathway modulators can be formulated into a pharmaceutically acceptable composition for administration to a mammal having atherosclerosis, at risk of developing atherosclerosis, or having had atherosclerosis, to treat the atherosclerosis (e.g., to reduce or reverse atherogenic plaque instability, to reduce the risk or rate of atherosclerosis progression, to reduce the risk of atherosclerosis reoccurrence, to increase the thickness of a fibrous cap over an atherosclerotic plaque, and/or to promote repair of a fibrous cap over an atherosclerotic lesion). For example, one or more IGF-1 signaling pathway modulators can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in a pharmaceutical composition described herein include, without limitation, saline, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol (PEG; e.g., PEG400), sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, and wool fat.

In some cases, when a composition containing one or more IGF-1 signaling pathway modulators is administered to a mammal having, at risk of developing, or having had atherosclerosis, the composition can be designed for oral or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intradermal) administration to a mammal. Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. In some cases, a composition containing one or more IGF-1 signaling pathway modulators can be formulated for administration by subcutaneous injection (e.g., subcutaneous injection to the abdomen, thigh, or upper arm).

A composition containing one or more IGF-1 signaling pathway modulators can be administered to a mammal having, at risk of developing, or having had atherosclerosis in any appropriate amount (e.g., dose). Effective amounts can vary depending on the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents (e.g., lipid-lowering agents), and the judgment of the treating clinician. An effective amount of a composition containing one or more IGF-1 signaling pathway modulators can be any amount that can treat a mammal having, at risk of developing, or having had atherosclerosis (e.g., can reduce atherosclerotic lesion size or reduce the progression rate of atherosclerosis) without producing significant toxicity to the mammal. For example, an effective amount of a IGF-1 signaling pathway modulator can be from about 0.01 to about 50 mg per kg of body weight, more preferably from about 0.1 to about 5 mg per kg of body weight. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the atherosclerosis in the mammal being treated may require an increase or decrease in the actual effective amount administered.

A composition containing one or more IGF-1 signaling pathway modulators can be administered to a mammal having, at risk of developing, or having had atherosclerosis at any appropriate frequency. The frequency of administration can be any frequency that can treat a mammal having, at risk of developing, or having had atherosclerosis (e.g., to reverse plaque instability, to reduce the risk or rate of atherosclerosis progression, to reduce the risk of atherosclerosis reoccurrence, to increase the thickness of a fibrous cap over an atherosclerotic plaque, to promote repair of a fibrous cap over an atherosclerotic lesion, and/or to reduce the size of an atherosclerotic lesion) without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a day to about once a week. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and route of administration may require an increase or decrease in administration frequency.

A composition containing one or more IGF-1 signaling pathway modulators can be administered to a mammal having, at risk of developing, or having had atherosclerosis for any appropriate duration. An effective duration for administering or using a composition containing one or more IGF-1 signaling pathway modulators can be any duration that can treat a mammal having, at risk of developing, or having had atherosclerosis (e.g., to reverse plaque instability, to reduce the risk or rate of atherosclerosis progression, to reduce the risk of atherosclerosis reoccurrence, to increase the thickness of a fibrous cap over an atherosclerotic plaque, to promote repair of a fibrous cap over an atherosclerotic lesion, and/or to reduce the size of an atherosclerotic lesion) without producing significant toxicity to the mammal. For example, the effective duration can vary from several months to several years or to a lifetime. In some cases, the effective duration can range in duration from about six weeks to about 12 months. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and route of administration.

In some cases, a course of treatment can be monitored. In some cases, methods described herein also can include monitoring the severity or progression of atherosclerosis in a mammal. Any appropriate method can be used to monitor the severity or progression of atherosclerosis in a mammal. For example, one or more symptoms of atherosclerosis can be assessed using any appropriate methods and/or techniques, and can be assessed at different time points. Techniques that can be used to diagnose and assess atherosclerosis include, without limitation, blood tests for cholesterol and sugar levels, ultrasound, ankle-brachial index, stress test, angiogram, ECG, CT scan, and MRA. In some cases, the methods described herein also can include monitoring a mammal being treated as described herein for toxicity. The level of toxicity, if any, can be determined by assessing a mammal’s clinical signs and symptoms before and after administering a known amount of a particular composition. It is noted that the effective amount of a particular composition administered to a mammal can be adjusted according to a desired outcome as well as the mammal’s response and level of toxicity. In some cases, the thickness of a fibrous cap can be measured using intravascular OCT, before and/or after treatment.

This document also provides methods and materials that can be used to identify agents that increase atherogenic plaque stability. For example, such a method can include measuring the thickness of a fibrous cap of an atherogenic plaque, contacting the atherogenic plaque with a candidate agent, and measuring the thickness of the atherogenic plaque after the contacting, to determine whether there has been a change in thickness of the fibrous cap. If the thickness of the fibrous cap increased, then the candidate agent can be identified as an agent that increases atherogenic plaque stability. Any suitable agent can be tested using the methods provided herein. For example, a candidate agent can be an IGF-1 receptor activator (e.g., an IGF-1 polypeptide, or a nucleic acid construct encoding an IGF-1 polypeptide), or an IGFBP-3 inhibitor (e.g., an IGFPB3 antisense oligonucleotide, or a nucleic acid construct encoding an IGFBP-3 neutralizing antibody).

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1 - Materials and Methods

Animal Models : C57BL/6 Ld!r mice were purchased from the Jackson Labs (stock number 002207) and interbred to generate homozygous Ld!r female mice for experiments. All mice were housed in a specific-pathogen free (SPF) facility with a 12 hour light/dark cycle and ad libitum access to food and water for the duration of experiments. Atherosclerosis Induction. C57BL/6 Ld!r female mice were raised to ~10 weeks of age on standard irradiated non-atherogenic chow diet (LabDiet #5053; 13.205% calories from fat), at which point they were enrolled to one of several experimental designs. HFD (TD. 88137; 42% calories from fat) was obtained irradiated from Harlan- Teklad, stored at 4°C until use, and exchanged weekly. In all cases, refers to the above standard non-atherogenic chow diet.

To analyze the impact of IGF- 1 on inner aortic curvature atherogenic lesions, female Ldlr mice were placed on HFD for 33 days and administered recombinant LR3 IGF-1 (ProspecBio, cat. no. CYT-022) by IP injection (0.3 mg/kg) in 10 mmol/L HC1/PBS Veh on a 5-days-on/2-days-off schedule for 3 weeks, and then daily for the final 10 days. During the last 5 days, EdU (50 mg/kg) was co-administered with IP LR3 IGF-1 or Veh (10 mmol/L HC 1/PBS) injection.

Blood Analysis. No more than 24 hours prior to sacrifice, blood was collected using retro-orbital sinus puncture with a heparinized glass capillary into an EDTA-treated microcentrifuge tube. Whole blood was immediately subjected to gross circulating cell analysis using the Hemavet 950 instrument (Drew Scientific Inc., Miami Lakes, FL). Plasma was isolated by centrifugation in EDTA-treated microcentrifuge tubes for 15 minutes at 4°C and 3500 g. Lipid analysis was performed using high-performance liquid chromatography.

SA -b-Gal Staining and GAL-EM: S A-b-Gal staining was performed using a kit (Cell Signaling; cat. no. 9860S) per the manufacturer’s instructions with slight modification. Briefly, whole aortas were excised, opened lengthwise, and stored in PBS on wet ice until fixation, then fixed for 15 minutes at room temperature (RT) in kit fixative. Aortas were then washed twice for 5 minutes in ice-cold PBS to remove excess fixative and transferred to pH 6.0 SA-P-Gal staining solution at 37°C for 12 hours.

SA b-Gal stained aortas intended for routine histological analysis were post-fixed in 10% neutral buffered formalin for 12 hours at RT before inner-curvature aortic arch plaque microdissection, standard dehydration, paraffin infiltration, embedding, and sectioning. SA b-Gal stained aortas intended for GAL-EM analysis were post-fixed in Trump’s fixative for 4 hours at RT. The inner-curvature aortic arch plaque was then microdissected and processed for electron microscopy (EM) by dehydration through xylene-alcohol series, followed by osmium-tetroxide counterstaining, infiltration with Epon resin, and sectioning. Between 1 and 4 plaque-bearing thin sections were analyzed per mouse for all plaque parameters. Cells were considered X-Gal+ if they contained one or more needle-shaped or cuboidal electron-dense crystals within a vacuole.

Cell-type identification was as described elsewhere (Childs et al, Science 354:472-477, 2016) and based on ultrastructural morphology. Briefly, approximately circular cells with numerous vacuolations were considered to be foam cell macrophage like. Spindle shaped or highly ramified cells with electron-dense cytoplasm rich in endoplasmic reticulum, stress fibers, and with few vacuoles were considered to be VSMC-like.

Immunohistochemical and Immunofluore scent Labeling : Immunohistochemical detection of Sma was performed using standard techniques. Briefly, 5 pm thick paraffin sections were rehydrated and antigen retrieval performed using high pH antigen unmasking solution (Vector, H-3301) before blocking with 2% normal goat serum and incubating overnight in primary antibody (mouse-anti-Sma (1:200; Abeam, cat. no. ab7817)) in 2% serum block at 4°C in a humidified chamber. Sections were then washed, incubated with biotin-conjugated goat-anti-rabbit secondary, washed, incubated with avidin-conjugated horseradish peroxidase enzyme, and developed with DAB kit (Vector, SK-4100). Sections were counterstained with dilute haemotoxylin. IHC staining for IGFBP-3 was performed as above, with the exception of retrieval in low pH antigen unmasking solution (Vector, H-3300) and incubating overnight with rabbit-anti-IGFBP-3 (1:200; Abeam, cat. no. ab76001).

For immunofluore scent co-labeling of Vim and Sma, 5 pm thick paraffin sections from SA b-Gal stained intermediate or late-disease remodeling mice were deparaflfmized by two 25-minute incubations in 50°C xylene before standard rehydration to 70% EtOH, 5 minute incubation in 0.85% NaCl/dH O, and transfer to PBS. Sections were then pre fixed in 4% PFAfor 7.5 minutes; antigen retrieved via Proteinase K (10 pg/mL) for 10 minutes; post-fixed in 4% PFA for 5 minutes; blocked with mouse-on-mouse blocking reagent (Vector Labs; cat. no. MKB-2213) for 30 minutes; blocked with 2% normal goat serum (NGS) for 30 minutes; and incubated in 1:150 Sma (Abeam, cat. no. ab7817) and Vim (Abeam, cat. no. ab92547) primary antibodies overnight at 4°C before secondary staining. Samples were Hoechst stained, washed, and mounted in Vectashield with 1:100 Hoechst to highlight both nuclei and retain Hoechst staining of elastic fibers. For all experiments, mice were stained and imaged in parallel on identical exposure settings, and at least 2 sections separated by at least 200 pm were assessed. IF SI is the space between the first pair of elastic fibers as identified by Hoechst; IFS2, the second pair; and, IF S3, the third pair. For IFS1 scoring, all cells beneath plaque on all sections were scored. For IFS2 and IFS3, at least one section from all mice was scored for all such cells. For flanking region scoring, no more than 20 IFSl nuclei beyond the plaque edge were assessed from 1-3 sections per mouse.

For Sma ⁺ scoring of fibrous cap, all fibrous cap cells throughout 2-3 sections separated by 200 pm or more were scored and normalized to the length of the total plaque luminal surface. Immunofluorescene (IF) quantifications of Sma content were performed on Sma/TUNEL costained material (see Histological Analyses section for protocol).

Histological Analyses :

Fibrous cap thickness measurements: Cap thickness was measured on routine H-E stained 5 pm-thick paraffin sections using 20x images and Image J software. The fibrous cap was operationally defined as largely uninterrupted strip of eosinophilic material with a higher density of nuclei than the plaque core. A single foam cell macrophage or small cholesterol crystal cleft was not considered to be a disruption and were included in measurements. At least 25 equally dispersed measurements of fibrous cap thickness per section were taken. Per mouse, between one and four sections were measured. Each plaque-bearing section thus measured was separated from others from the same mouse (if applicable) by 200 pm. Multiple sections were averaged to produce per-mouse fibrous cap thickness measurements where applicable. By TEM, fibrous cap was defined as a region of cells with VSMC morphology interrupted by no more than a single cell with foam cell macrophage morphology. At least 20 length measurements were taken per section for both H-E and TEM quantifications. Quantification of cells traversing the innermost elastic lamina: Briefly, images of all elastic fiber breaks per section for at least two H-E-stained plaque sections per mouse were measured for the length between elastic fiber free ends and used to calculate the average length of disrupted innermost elastic fiber per break. Total nuclei intersecting a straight line joining each pair of free fiber ends were then counted. The number of medial cell nuclei crossing the first fiber was then normalized to total gap length. Sections with no breaks and no cells crossing the first elastic lamina were considered as “0 cells/pm of break length,” but were not considered in the average break length calculation. For TEM- based measurements, total fiber crossing cells per plaque-bearing section were assessed by inspecting the first elastic fiber at IO,OOOc magnification. Between one and four sections per mouse were assessed for fiber crossing cells for all mice so assessed.

Verhoeff-Van Gieson-positive fine elastin fibers: Density of elastin fine fibers was measured on 5 pm-thick paraffin sections stained with Verhoeff-Van Gieson per the manufacturer’s protocol (Polyscientific R&D, cat. No. k059). Using a lOOx oil immersion lens, all Verhoeff-Van Gieson fine fibers of any length were manually counted across the entire fibrous cap and then normalized to total length of the lumen-facing surface of the lesion. One to three sections separated by 200 pm (if applicable) were used to produce per-mouse elastin fine fiber density measurements.

Alizarin red staining: Staining was performed on 5 pm thick paraffin sections. Briefly, slides were rehydrated through xylene-ethanol-water gradient, stained for 2 minutes in 2% alizarin red S/dEEO solution, cleared with acetone, and dehydrated in 1:1 mixture of acetone and xylene, before mounting in CYTOSEAL™. Bright red-orange material was considered alizarin red-positive, and normalized to total neointimal area. At least two sections separated by 200 pm or more were scored and averaged to derive per- mouse alizarin-red positive area measurements.

TUNEL staining: Paraffin sections of plaque were stained for TUNEL positivity according to the manufacturer’s instructions (DeadEnd fluorometric TUNEL kit, Promega, cat. no. G3250; in situ cell death detection kit, fluorescein, Roche, cat. no. 11684795910), followed by overnight staining for Sma at 4°C (Abeam, mouse, 1:250). A cell was considered TUNEL ⁺ if bright TUNEL signal overlapped completely with Hoechst nuclear counterstain.

EdU staining: Paraffin sections of plaque were stained for EdU positivity according to the manufacturer’s instructions (Click-iT EdU cell proliferation kit, Thermo- Fischer, cat. no. C10337 Alexa-488 conjugated) followed by overnight staining with anti- Sma at 4°C (Abeam, mouse, 1:150) or anti-Sma/anti-tdTom (1:150 and 1:200, respectively). One to two sections per mouse separated by at least 200 pm or more were analyzed for frequency of Sma ⁺ /EdU ⁺ cells as the percentage of total Sma ⁺ or Sma ⁺ /tdTom ⁺ cells throughout the lesion.

Generation of Conditioned Media: MEFs were generated at embryonic day 13.5. Cells were expanded in DMEM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine, non-essential amino-acids, sodium pyruvate, gentamicin and b-mercaptoethanol, at 3% O2 and used for experiments at passage (P)5. To establish irradiated-senescent MEFs, subconfluent cells were exposed to 10 Gy g-radiation ( ¹³⁷ Caesium source) and cultured for 10 days. Confluent cell layers of irradiated SNCs (IR-SNCs) and replication- competent (nonSNC) controls were washed twice with PBS before addition of 5 mL of DMEM/F12 (0.5% FBS) per T75. After 48 hours of conditioning, medium was harvested, filtered through a 0.2 pm syringe filter, aliquoted and stored at -80°C until use.

Neutralizing Antibody Preparation : For experiments in FIGS. IB, 1C, 3B, 3D, and 2B, polyclonal rabbit IgG control (Abeam, cat. no. AB 171870) and polyclonal rabbit a-IGFBP-3 Ab (Abeam, cat. no. AB76001) were stored at 4°C until use. To avoid freeze/thaw cycling and remove stabilizing excipients such as azides, prior to application to cultured tissue or cells, an appropriate quantity of antibody was washed 5 times with a 5x volume of sterile PBS using 5 kD size-exclusion columns (Vivaspin 500, GE Healthcare, cat. no. 38-9322-23) in a 4°C microcentrifuge (12,000 RCF, 15 minutes), resuspended in PBS to a final concentration of 1 pg/mL, and stored at 4°C until use for no more than 24 hours. For experiments in FIG. 3E, recombinant a-IGFBP-3 Ab (Abeam, cat. no. AB224530), rabbit a-IGFBP-3 Ab (Fisher, cat. no. PA5-29711), or polyclonal rabbit IgG control (Abeam, cat. no. AB 171870) were purified as above on receipt, and stored in individual aliquots at -20°C to avoid freeze-thaw effects.

Human VSMC Migration Assays: Primary, normal human aortic VSMCs were obtained from the American Type Culture Collection (cat. no. ATCC PCS-100-012; female donor) and expanded for 4 passages in vascular cell basal media (cat. no. ATCC PCS-100-030) supplemented with growth factors (VSMC growth kit; cat. no. ATCC PCS-100-042). All experiments were done on VSMCs between P4 and P10.

For scratch wound assays of VSMC migration, 40 x 10 ³ VSMC were seeded per well in fresh VSMC basal media on a 24-well tissue culture plate and grown to confluence for 96 hours. AP20 pipette tip was used to make a single linear scratch across the well. Culture medium was immediately changed to 150 pL fresh VSMC basal media, to which was added an equal volume of either IR-SNC CM with polyclonal rabbit IgG control (Abeam, cat. no. AB 171870), nonSNC CM with polyclonal rabbit IgG control, IR-SNC CM with rabbit a-IGFBP-3 Ab (Abeam, cat. no. AB76001), or IR-SNC CM with recombinant LR3 IGF-1. At least three random 20x fields per scratch wound were analyzed at 4 post-scratch for VSMC immigrating into the wound space, for a total of 6 scratch wounds per condition analyzed. Each wound was treated with CM derived from a distinct T75 of MEFs cultured per the Generation of Conditioned Media methods subsection. The end concentration of all antibodies was 4 pg/mL, and the end concentration of LR3 IGF-1 was 120 ng/mL.

For IF co-staining of Sma and Vim, 1.8 x 10 ³ VSMC were seeded per well on glass chamber slides in VSMC basal media and allowed to adhere for 12 hours. The culture medium was then exchanged for 20 hours with 200 pL 50% fresh VSMC basal media and 50% one of the following: IR-SNC CM with polyclonal rabbit IgG control (Abeam, cat. no. AB 171870), nonSNC CM with polyclonal rabbit IgG control, IR-SNC CM with rabbit a-IGFBP-3 Ab (Abeam, cat. no. AB76001), or IR-SNC CM with recombinant LR3 IGF-1. The end concentration of all antibodies was 4 pg/ml, and the end concentration of LR3 IGF-1 was 120 ng/mL. Cells were fixed for 12 minutes in 4% PFA/PBS, permeabilized in 0.5% Triton X-100/PBS for 5 minutes at room temperature, and stained for 30 minutes at room temperature for Sma and Vim (1 :300). Using ImageJ, at least 50 cells from two fields per line of CM were scored for integrated signal density of Sma and Vim to generate a per-line average signal intensity. These intensity measurements were normalized to Div+IgG signal from 1-3 concurrently imaged lines.

Murine Aortic Ring Cultures. For aortic ring explant experiments, thoracic aortas from 6-week-old C57BL/6 female mice were briefly perfused with ice-cold PBS and then excised. ~1 mm thick rings were collected and transferred to PBS on wet ice. A 48-well plate was then prepared with wells containing 300 pL media (50% fresh DMEM/F12 (20% FBS) and 50% conditioned media (0.5% FBS), supplemented to obtain 20% FBS concentration during the assay, and either 5 pg rabbit IgG (Abeam, cat. no. AB 171870) or a-IGFBP-3 9Abcam, cat. No. AB76001). Conditioned media was an equal-volume mixture of CM from three T75 flask cultured per the Generation of Conditioned Media section. Aortic rings were transferred to this plate, and left to incubate undisturbed at 37°C in a 5% CO2 incubator. At 72 hours, the number of outgrowing VSMCs beyond each aortic ring’s circumference was manually counted at 40x magnification.

Murine Plaque Explant IGFBP-3-neutralization and Vim/Sma Colabeling.

Female Ld!r mice were fed HFD for 12 weeks, then switched to LFD for 2 weeks to normalize circulating lipid profile. After CO2 euthanasia, aortas were flushed 2x with ice- cold PBS, the aortic arch was removed, and ~1 mm thick cross-sectional rings of plaque bearing aortic arch were explanted to 125 pL DMEM/F12 with 0.5% FBS. Either 8 pg/mL of rabbit a-IGFBP-3 (Abeam, cat. no. AB76001) or polyclonal rabbit IgG control (Abeam, cat. no. AB 171870) antibody was added per well and incubated in a humidified chamber at 37°C/5% CO2 for 18 hours. Rings were then fixed at RT in 10% neutral buffered formalin for 12 hours and processed for paraffin embedding. One 5 pm thick paraffin section per ring was then stained for 12 hours at 4°C with 1 : 150 rabbit a- Vim and mouse a-Sma, imaged completely at 20x magnification, and the proportion of IFS1 cells with the indicated marker profile was determined manually by an operator blinded to sample identity.

Human Plaque Explant IGFBP-3-neutralization and Vim/Sma Co-labeling. Discarded endarterectomy tissue from either femoral or aortoiliac sites was obtained from patients undergoing therapeutic endartectomy. Tissue was collected into sterile saline and stored at 4°C for up to 1 hour until processing. Serial ~2-3 mm slices were prepared from the explanted material and transferred individually into 450 pL prewarmed DMEM/F12 with 0.5% FBS containing either (for FIG. 3D) 8 pg/mL of rabbit a-IGFBP-3 Ab (Abeam, cat. no. AB76001) or polyclonal rabbit IgG control (Abeam, cat. no. AB171870) or (for FIG. 3E) 8 pg/mL rabbit recombinant a-IGFBP-3 Ab (Abeam, cat. no. AB224530), rabbit a-IGFBP-3 Ab (Fisher, cat. no. PA5-29711), or polyclonal rabbit IgG control Abeam, cat. No. AB171870). Each a-IGFBP-3 treated explant slice was matched to at least one adjacent IgG-treated slice from the same patient and lesion. Samples were incubated in a humidified chamber at 37°C/5% CO2 for 48 hours and fixed at 4°C for 12 hours in 4% PFA/PBS. Samples were then decalcified by gentle inversion in 15 mL of 10% HCl/10% acetic aci d/80% dFFO for 12 hours at RT, post-fixed for 2 hours at 4°C in 4%PF A/PBS, and processed for paraffin embedding. 5 pm thick paraffin sections were then stained overnight (FIG. 3D) or for 1.5 hours at room temperature (FIG. 3E) with 1:150 rabbit a- Vim and mouse a-Sma. Due to high blue-channel autofluorescence, costaining with Hoechst was uninformative. Therefore, colocalization of goat-a-rabbit Alexa-488 and goat-a-mouse Alexa-594 was determined as % co-positive pixels per 20x field using the colocalization function in Cell SENS software. Briefly, an operator blinded to sample identity identified at least three 20x fields containing Sma ⁺ cells per section, which were then imaged for both Sma and Vim without considering Vim status. Both IgG- and a-IGFBP-3 -treated samples were stained and imaged in parallel. Between 1-3 explant slice pairs were analyzed per patient, to generate a per-patient % colocalization with IgG or a-IGFBP-3 treatment.

RNA-seq Library Preparation, Sequencing and Bioinformatic Analyses : RNA extraction (RNeasy Micro kit, #74004, Qiagen) was performed according to the manufacturer’s instructions. RNA quality and quantity were assessed using Agilent Bioanalyzer RNA 6000 Pico chips (#5067-1513, Agilent Technologies). About 100 ng high-quality RNA from microdissected plaques or 10 ng from microdissected individual inner curvature of aortic arch were subjected to library preparation using the TruSeq RNA Library Prep Kit v2 (#RS- 122-2001, Illumina), according to the manufacturer’s instructions. The concentration and size distribution of the completed libraries were confirmed using Agilent DNA 1,000 chips (#5067-1504, Agilent Technologies) and Qubit fluorometry (Qubit dsDNAHS, #Q32851, Invitrogen). Libraries were sequenced following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. Flow cells were sequenced as 100 X 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS 3.3.20 collection software. Base calling was performed using Illumina’s RTA2.5.2 software. RNA sequencing was performed at the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility (Mayo Clinic, Rochester, Minnesota). Fastq files of paired-end reads were aligned with Tophat 2.0.14 to the UCSC reference genome mmlO using Bowtie22.2.6 with default parameters. Gene level counts were obtained using FeatureCounts 1.4.6 from the SubRead package with gene models from corresponding UCSC annotation packages. Differential expression analysis was performed using R package DESeq2 1.10.1 after removing genes with average raw counts less than 10. Genes with false discovery rate (FDR) < 0.05 irrespective of unshrunk log2 fold change as determined by DESeq2 were considered significantly upregulated or downregulated. Heatmaps were generated with Morpheus, Broad Institute (software.broadinstitute.org/morpheus) using lfcMLE values and negative Log 10 (FDR) values.

RT-qPCR analysis. RNA was extracted from pulverized brachiocephalic arteries (aortic insertion through bifurcation) using a RNeasy Micro kit (Qiagen, cat. no. 74004). cDNAwas synthesized from 1 ng RNA per reaction using a Superscript III 1st Strand cDNA synthesis kit (ThermoFisher, cat. no. 18080051). RT-qPCR was performed using SYBR Green PCR master mix (Applied Biosystems, cat. no. 4309155) according to the manufacturer’s protocol and normalized to GAPDH expression. Primers used were: IGFBP-3 (fwd): 5 '-TAAGAAGAAGC AGTGCCGCC-3 ' (SEQ ID NO:4)

IGFBP-3 (rev): 5'-TTTCCCCTTGGTGTCGTAGC-3' (SEQ ID NO:5)

Igfbp2 (fwd): 5'-GGGTGCCAAACACCTCAG-3 ' (SEQ ID NO:6)

Igfpb2 (rev): 5 '-AGGTTGTACCGGCCATGC-3 ' (SEQ ID NO:7)

Igfbp5 (fwd): 5'- ATACAACCCAGAACGCCAGCT-3 ' (SEQ ID NO:8)

Igfbp5 (rev): 5'- ACCTGGGCTATGC ACTTGATG-3 ' (SEQ ID NO:9)

Igfbp6 (fwd): 5'- CAACCCCGAGAGAACGAAGAG-3 ' (SEQ ID NO: 10) Igfbp6 (rev): 5'- TCTCCTTTGTAGTCTCCTCCG-3' (SEQ ID NO: 11)

GAPDH (fwd): 5'-TGCACCACCAACTGCTTA-3' (SEQ ID NO: 12)

GAPDH (rev): 5 '-TGGATGC AGGGATGATGTTC-3 ' (SEQ ID NO: 13) Statistical Analyses : All statistical analyses were performed using Graphpad Prism software, version 8.0.

Example 2 - SNCs suppress innate smooth muscle repair in atherosclerosis The studies described herein utilized the Ldlr ^f mouse model, which develops HFD-induced dyslipidemia and atherosclerosis. To mimic the clinically-relevant context of atheromas subject to medical management of proatherogenic dyslipidemia, an approach was used in which HFD-fed Ldlr mice were switched to a LFD following establishment of atherosclerosis, thereby dramatically reducing circulating pro atherogenic lipids.

IGFBP-3 inhibits VSMC promigratory switching

To understand how lesional SNCs inhibit recruitment of cap-forming medial VSMCs, a potential role for insulin-like growth factor binding protein 3 (IGFBP-3) was investigated. Consistent with the idea that IGFBP-3 is a senescence associated secretory phenotype (SASP) factor in the suppression of VSMC migration, IHC for IGFBP-3 uncovered that a very high proportion of SA b-GaE lesional cells stained positive for the IGF inhibitor (FIG. 1A). A subset of SA b-Gal- lesional cells was also IGFBP-3 ⁺ (FIG. 1A).

To probe the role of the SASP and its constituent IGFBP-3 in inhibiting VSMC migration, two established in vitro migration assays were used: outgrowth of VSMCs from ex vivo cultured aortic rings, and scratch wounding of confluent VSMC cultures

(Zhang et al, Arteriosclerosis Thrombosis Vase Biol 25:533-538, 2005; Yang and

Proweller, J Biol Chem 286:13741-13753, 2011; and Nicosia, J Cell Mol Med 13:4113-

4136, 2009). In the first assay, conditioned medium from irradiated senescent-MEFs (IR-

SNC CM) reduced the number of outgrowing VSMCs surrounding murine aortic rings versus rings treated with CM from proliferating MEFs (nonSNC CM) (FIGS. IB and 2A). Importantly, addition of IGFBP-3 -neutralizing antibody to IR-SNC CM restored normal VSMC outgrowth. Likewise, in scratch wound assays, IR-SNC CM repressed emigration of human VSMCs into the bare wound space, but not in the presence of IGFBP-3 -neutralizing antibody (FIG. 1C), providing further evidence that SNCs act to inhibit VSMC migration through the SASP, with IGFBP-3 as an SASP factor. Two additional observations supported this mechanism. First, supplementation of IR-SNC CM with recombinant LR3 IGF-1, a potent IGF-1 variant with reduced Igfbp binding affinity (von der Thusen et al, supra) normalized VSMC migration (FIG. 1C). Second, IR-CM increased Sma levels and suppressed VIM expression of cultured human VSMCs, indicative of synthetic-to-contractile switching, a phenomenon prevented by the presence of LR3 IGF-1 or IGFBP-3 -neutralizing antibody (FIGS. 2B and 2C).

This mechanism was further investigated in vivo by administering LR3 IGF-1 or Veh to Ldlr ^ mice during a 33-day HFD feeding period (FIG. ID). Examining lesions in the aortic arch inner curvature, it was observed that the number of Sma ⁺ VSMCs in the sub endothelium was two-fold higher in LR3 IGF-1 -treated mice than in Veh-treated counterparts, which correlated with an increase in cells crossing the first fiber (FIGS. IE and IF). LR3 IGF-1 treatment did not affect lesion size or overt SA b-Gal activity (FIG. 1G)

IGFBP-3 inhibits medial VSMC dedifferentiation in murine and human plaque explants

Next, the role of IGFBP-3 in suppressing VSMC promigratory phenotype switching was investigated using ex vivo culture of atherosclerotic plaques (Lebedeva et al, Atherosclerosis 267:90-98, 2017). Plaque-rich aortic arches were collected from Ldlr mice that were fed HFD for 12 weeks and then placed on LFD for 2 weeks. Rings prepared from these arches were cultured for 18 hours in the presence of anti-IGFBP-3 or IgG control antibody (FIG. 3A). Immunolabeling of sections prepared from these explants revealed that IGFBP-3 -neutralizing antibody markedly increased the number of Vim ⁺ /Sma cells in IFS1 beneath plaques while simultaneously reducing the number of Vim /Sma ⁺ cells (FIG. 3B), further supporting the conclusion that IGFBP-3 is involved in suppressing lesional recruitment of medial VSMCs. To assess the potential applicability of these findings to human atherosclerosis management, aortic and femoral endarterectomy specimens were collected and prepared as paired serial slices from these lesions, and then cultured in the presence of IgG or one of three distinct IGFBP-3- neutralizing antibodies for 2 days (FIGS. 3C-3E). IGFBP-3 neutralization increased the number of SMA ⁺ cells expressing VIM in human atheroma explants, regardless of their site of origin (FIGS. 3D and 3E), implying that a mechanism by which SNCs suppress VSMC synthetic switching in murine plaques may be conserved in human disease tissue.

Despite effective medical management of dyslipidemia, patients with advanced atherosclerosis face significant residual risk of death or disability from fibrous cap disruption (Bittencourt and Cerci, supra ; and Reith and Armitage, supra). The results presented herein indicate that SNCs in atheromas act through a lipid-independent mechanism: antagonism of IGF-1 mediated pro-migratory VSMC contractile-to-synthetic phenotypic switching in the aortic wall through the paracrine actions of a core S ASP component, IGFBP-3, thereby impairing fibrous cap formation, maintenance, and restoration (FIG. 4). From the earliest stages of atherogenesis onwards, SNCs inhibited dedifferentiation of VSMCs in the first interfiber space of the arterial wall underneath atherosclerotic plaque to a promigratory phenotype, thereby limiting entry of medial VSMCs into the lesion for fibrous cap assembly or maintenance. Mechanistic experiments revealed that IGFBP-3 produced by SNCs can antagonize IGF-1 -mediated promigratory VSMC phenotype switching in cultures of pure VSMCs, as well as murine and human plaque explants. Taken together, the results described herein demonstrated that SNCs can contribute to end-stage fibrous cap deterioration by suppressing intrinsic tissue repair processes. This mechanism is reversible (as evidenced by restored VSMC numbers, elastin content, and overall cap thickness), and is a determinant of plaque vulnerability in atherosclerosis, indicating that the intermittent use of IGF-1 signaling pathway modulators (e.g., to inhibit or neutralize IGFBP-3) in combination with lipid lowering drugs are therapeutically beneficial.

In summary, the results presented herein demonstrate that atherogenic plaque instability in a mammal (e.g., a human) can be reversed by administering an IGF-1 signaling pathway modulator (e.g., an IGF-1 receptor activator such as an IGF-1 polypeptide or an IGF-2 polypeptide, or an IGFBP-3 inhibitor such as an IGFBP-3 antibody) at an amount effective to increase a thickness of a fibrous cap of a thin-cap fibroatheroma. OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.