More specifically, the invention relates to novel animal models used in characterizing and researching proliferative diabetic retinopathy. Further, the invention relates to testing methods for retinopathic proliferation.

BACKGROUND OF THE INVENTION Diabetic retinopathy is a leading cause of blindness in humans. Pericyte loss is a hallmark of early diabetic retinopathy in humans and other mammals, but it is unclear if it constitutes a causal pathogenic event in the progression of the disease or is a phenomenon without further pathogenic implications. Diabetes in rodents causes up to 50% pericyte loss but leads only to mild microangiopathy ; progression into proliferative retinopathy and blindness does not occur. While the pericyte loss correlates with the formation of acellular capillaries and thickening of the capillary basement membrane, only rarely do microaneurysms develop in these animals. Thus, only the earliest signs of human retinopathy develop in diabetic rodents in spite of significant pericyte loss. Improved animal models exhibiting symptoms seen in later stages of human diabetic retinopathy are needed.

In the initial response to diabetes related hyperglycemia, the retinal vasculature develops a number of characteristic pathological features. These include pericyte loss as well as increased vascular permeability, increased numbers of acellular and occluded capillaries, microaneurysms, basement membrane thickening and capillary cell loss. In diabetic humans these changes do not impact vision. Progressive changes in the retinal vasculature involve increased vascular permeability leading to focal exudation and macular edema, and hemorrhage, which may significantly impair vision21.

The retinopathy may also develop into a proliferative stage in which new blood vessels grow into the vitreous. These vessels and associated connective tissue may eventually cover the retina, causing blindness. Since the vascular changes in diabetic retinopathy are temporally correlated, the general assumption has been that a linear and causal chain of events leads from early to late stages of diabetic retinopathy. Actual causal links, however, have not been established. This may partly be due to lack of effective animal models useful in studying progression or later stages of retinopathy.

Multiple proposals exist to explain how hyperglycemia results in progressive vascular change. Increased glucose concentration may trigger intracellular events such as increased polyol-and hexosamine pathway flux, protein kinase C (PKC) activation and intracellular formation of advanced glycation end-products (AGE). These events may be linked through a common denominator, the increased production of superoxide by the mitochondrial electron transport chain24. Increased oxidative stress and associated cellular metabolic changes may underlie the early microvascular abnormalities associated with diabetes. Well tailored research models would be helpful in resolving the multiple unanswered questions in this area.

The earliest morphological sign of diabetes-induced vascular abnormalities is pericyte loss from retinal capillaries. Pericyte loss has been considered a prerequisite for microaneurysm formation by generating local weakenings and subsequent outpouchings of the capillary wall. Pericyte loss also may be linked to the formation of acellular capillaries, since pericyte coverage has been correlated with endothelial survival in tumors. As the early stages of diabetic retinopathy involve pericyte loss, it would be an advancement in the state of the art to know if pericyte loss actually constitutes a causal event in retinopathy pathogenesis, or if it is a secondary consequence unrelated to disease progression.

All capillaries are partially covered by mural cells of the vascular smooth muscle cell (vSMC) lineage, known as pericytes, whose actual function is largely unknown. Pericytes make intimate contact with the endothelium throughout the central nervous system (CNS).

The abundance, distribution and association of pericytes with the endothelium varies in different microvascular beds, with the highest abundance of pericytes in retinal capillaries.

In the kidney glomerulus and the mouse placenta labyrinthine layer, the pericytes, called mesangial cells in glomeruli, form matrix-laden core structures around which capillary loops organize. At these sites, pericytes appear to promote the splitting of vessels into dense capillary tufts during development, reviewed in Betsholtz et al2.

Pericytes have also been proposed to stabilize blood vessels in the retina3 and in tumors4. Targeted disruption of platelet-derived growth factor B (PDGFB) and PDGF P- receptor (PDGFRß) genes in mice5 6 show that PDGFB/PDGFRß signaling is critical for pericyte recruitment in angiogenesis7 8. Absence of pericytes in these mice leads to severe vascular dysfunction, manifested as lethal hemorrhage, stasis, microaneurysms and edema.

Pericyte-deficient vessels or capillaries are irregular in diameter, leaky, display endothelial hyperplasia, abnormal endothelial ultrastructure and irregular distribution of intercellular junctions9. Since, according to the law of Laplace, vessel wall tension is proportional to vessel wall radius at constant blood pressure, focal dilations may rapidly expand to microaneurysms. Numerous rupturing microaneurysms develop in late gestation of pericyte- deficient PDGFB and PDGFRß null mice. These vascular lesions are reminiscent of those seen in human diabetic microangiopathy, providing support for a causal role of pericyte loss in pathogenesis of the disease, but failing to provide a viable animal model that will survive gestation.

Additional murine knockout mutants, such as those lacking angiopoietin-1/Tie-2, TFG-ß, and EDG-1, have also been reported to be variably pericyte and/or vSMC deficient.

These mutants are also lethal prior to birth due to cardiovascular dysfunction3l~36. TGF-ß signaling appears to be critical for early vSMC/pericyte formation, whereas PDGF-B/-R-P and EDG-1 signaling seems to be involved in later proliferation and migration of these cells.

Angiopoietin-1 primarily targets Tie-2 receptors on endothelial cells and its role in vSMC/pericyte formation might therefore be indirect.

The microvascular abnormalities caused by pericyte deficiency in PDGFB and PDGFRp mutants are similar to those seen in diabetic microangiopathy in humans. The retinal vascular bed in human diabetic subjects displays microaneurysms, focal exudation and microhemorrhage. While these changes do not severely compromise vision, progression may take place into proliferative retinopathy, a severe and irreversible condition characterized by massive cell proliferation and angiogenic sprouting into the vitreous, leading to blindness.

Upregulated expression of glial fibrillary acidic protein (GFAP) in radial Müller glia is one of the earliest cellular abnormalities reported to occur in the retina of diabetic rats25.

Pericyte loss is the earliest morphological sign of a vascular abnormality in diabetic patients. In determining whether pericyte loss is a causal or secondary event in retinopathy pathogenesis, attempts have been made to utilize animal models. The phenotype of PDGFB and PDGFRp mutants provides conceptual support for a causal role between pericyte loss and retinopathy pathogenesis, however, data from diabetic mice and rats conflict with this view.

Retinal capillaries in such animals loose up to 50% of their pericytes, but as noted above, only mild microangiopathy develops. Microaneurysms are rarely seen and progression into proliferative retinopathy has never been reported. Pericyte loss in PDGFB and PDGFRp mutants is more extensive than in typical rodent diabetes, but consequences in the retina have not been possible to address because these animals die before retinal vessels develop. It would be advantageous to have a way to ablate pericytes that is compatible with postnatal life and therefore provide improved study models for researchers.

SUMMARY OF THE INVENTION In the present invention, viable animal models were created to bypass diabetes as a cause of pericyte loss in the retina.

According to a first embodiment of the present invention, a method of creating a retinopathy or microangiopathy research organism is provided, comprising inactivating PDGF-B within a DNA segment of an organism, transferring the inactivated DNA into a blastocyst of an organism, transferring the blastocyst into a synchronized recipient female organism to produce a pregnant organism, and allowing gestation in the pregnant organism to proceed for a period of time sufficient to allow the development of a viable retinopathy or microangiopathy research organism. Optionally, the DNA may be inactivated by nucleotide deletion, nucleotide substitution, an antisense sequence, RNA interference, inhibiting antibodies, inhibiting affibodies, use of PDGF-B receptor antagonists or use of soluble ligand binding fragments of receptor. Optionally, the DNA segment may be endothelial cell DNA.

Optionally, the organism may be a non-human mammal, for example, a mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig or monkey.

According to a further embodiment of the present invention, a retinopathy or microangiopathy research organism is provided, comprising an organism with an inactivated gene encoding PDGF-B protein. Optionally, the organism may have an inactivated exon 4 of the gene encoding PDGF-B protein, the inactivation optionally accomplished by deletion of exon 4. Optionally, the research organism may be a non-human mammal, for example, a mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig or monkey.

According to a further embodiment of the present invention, a method of evaluating a potential therapy for retinopathy is provided, comprising administering the potential therapy to a research organism according to the previous embodiment and evaluating the research organism for change in retinopathic progression, wherein a decrease or slowing in retinopathic progression indicates the potential therapy is beneficial. Optionally, the method may further comprise administering a diabetes inducing agent to the research organism.

According to a further embodiment of the present invention, a method of creating a retinopathy or microangiopathy research organism is provided, comprising inactivating PDGF-B within a DNA segment of an organism, transferring the inactivated DNA into a blastocyst of an organism, transferring the blastocyst into a synchronized recipient female organism to produce a pregnant organism, allowing gestation in the pregnant organism to proceed for a period of time sufficient to allow the development of a viable offspring, and mating the offspring with a diabetic research organism to produce a retinopathy or microangiopathy research organism. Optionally, the diabetic research organism may be a leptin deficient organism.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 schematically represents a partial map of the PDGF-B wild type initial recombinant, floxed and lox alleles and the targeting construct, where boxes indicate exons, E4 stands for exon 4, Neo represents a PGK-neo cassette, R represents an EcoRl site and triangles denote loxP sites; Figure 2 shows southern blot analysis results of genomic EcoRI digests; Figure 2A distinguishes wild type and recombinant alleles; Figure 2B shows discrimination between genomes with recombinations on either side of the 3'loxP site; Figure 2C shows a conversion from recombinant to flox allele; Figure 3 schematically represents the PCR strategy used to discriminte between wild type, knock out (K/0), flox, and lox alleles; Figure 4 shows a three primer PCR discriminating between wild type, flox and lox alleles in DNA prepared from brain microvascular fragments, where numbers 1-9 represent fragment preparations from different lox/+ individuals; Figures 5A-8B utilize-galactosidase stainings to reveal pericyte nuclei; Figure 5A shows pericytes in a brain sample from a control organism at day E15. 5, where the rectangle indicates the location and orientation of the sample analyzed for Figure 5B, where fb represents forebrain and mb represents midbrain; Figure 5B shows pericyte distribution along vessels in midbrain samples, where men represents meningeal surface, vent represents ventricular surface, Rad br represents radial vascular branches and SVZ represents subventricular zone; Figure 6A shows pericytes in a brain sample from a PDGF-B-/-genotype organism at day E15. 5; Figure 6B shows pericyte distribution along vessels in midbrain samples taken from the PDGF-B-/-organism of Figure 6A; Figure 7A shows pericytes in a brain sample from a first PDGF-B lox/-genotype at day E15. 5; Figure 7B shows pericyte distribution along vessels in midbrain samples taken from the PDGF-B lox/-organism of Figure 7A, where arrows indicate radial vessels with normal pericyte abundance and arrowheads indicate radial vessels without pericytes; Figure 8A shows pericytes in a brain sample from a second PDGF-B lox/-genotype organism at day E15. 5; Figure 8B shows pericyte distribution along vessels in midbrain samples taken from the second PDGF-B lox/-organism of Figure 8A; Figure 9 is a side view of a 21-day-old wild type (left) and a 21-day-old PDGF-B lox/- (right) mouse brain stained to reveal pericytes; Figure 10A shows a surface view of a wild type organism, where A indicates artery and V indicates vein; Figure 10B shows a surface view of a sample from a wild type organism showing meningeal vessels; Figure 10C shows an enlarged surface view of a wild type organism brain sample; Figure 11 shows a midline view of a 21-day-old wild type (left) and PDGF-B lox/- (right) mouse brain stained to reveal pericytes; Figure 12A shows a surface view of a 21-day-old PDGF-B lox/-mouse, where A indicates artery and V indicates vein; Figure 12B shows a surface view of a sample from a 21-day-old PDGF-B lox/-mouse showing meningeal vessels, where the arrowheads indicate long stretches of pial arteric branches devoid of XlacZ4 positive cells; Figure 12C shows an enlarged surface view of a brain section from a 21-day-old PDGF-B lox/-mouse; Figure 13A shows the retina of a wild type organism stained to reveal pericytes; Figure 13B shows pericyte density and distribution in the retina of a wild type organism; Figure 13C shows pericyte density and distribution in the pial vascular plexus of a wild type organism; Figure 13D shows pericyte density and distribution in the cerebellum of a wild type organism; Figure 13E depicts the relative quantification of pericyte density at five distinct sites in and adjacent to the CNS of a wild type organism where PP is the pial plexus, FC is the forebrain cortex, TH is the thalamus, CE is the cerebellum, and RE is the retina; Figure 14A shows the retina of a first PDGF-B lox/-organism, which has greater than 50% of the normal pericyte density at CNS sites; Figure 14B shows pericyte density and distribution in the retina of the first PDGF-B lox/-organism; Figure 14C shows pericyte density and distribution in the pial vascular plexus of the first lox/-organism; Figure 14D shows pericyte density and distribution in the cerebellum of the first PDGF-B lox/-organism; Figure 14E depicts the relative quantification of pericyte density at four distinct sites in and adjacent to the CNS of the first PDGF-B lox/-organism; Figure 15A shows the retina of a second PDGF-B lox/-organism, which has greater than 50% of the normal pericyte density at CNS sites; Figure 15B shows pericyte density and distribution in the retina of a second PDGF-B lox/-organism; Figure 15C shows pericyte density and distribution in the pial vascular plexus of a second PDGF-B lox/-organism; Figure 15D shows pericyte density and distribution in the cerebellum of a second PDGF-B lox/-organism; Figure 15E depicts the relative quantification of pericyte density at four distinct sites in and adjacent to the CNS of a second PDGF-B lox/-organism ; Figure 16A shows the retina of a third PDGF-B lox/-organism, which has less than 50% of the normal pericyte density at CNS sites; Figure 16B shows pericyte density and distribution in the retina of a third PDGF-B lox/-organism, where the asterisk indicates a region of sparse pericyte density and the triangle indicates a region of increased pericyte density, and depicting retinal changes such as advanced vessel abnormalities and retinal traction; Figure 16C shows pericyte density and distribution in the pial vascular plexus of a third PDGF-B lox/-organism; Figure 16D shows pericyte density and distribution in the cerebellum of a third PDGF-B lox/-organism ; Figure 16E depicts the relative quantification of pericyte density at four distinct sites in and adjacent to the CNS of a third PDGF-B lox/-organism, where the upper portion of the RE bar, marked with a triangle, indicates regions of increased pericyte density and the asterisk denotes sparse regions with densities similar to the rest of the CNS; Figure 17A shows the retina of a fourth PDGF-B lox/-organism; Figure 17B shows pericyte density and distribution in the retina of a fourth PDGF-B lox/-organism, where the asterisk indicates a region of sparse pericyte density and the triangle indicates a region of increased pericyte density ; Figure 17C shows pericyte density and distribution in the pial vascular plexus of a fourth PDGF-B lox/-organism; Figure 17D shows pericyte density and distribution in the cerebellum of a fourth PDGF-B lox/-organism; Figure 17E depicts the relative quantification of pericyte density at four distinct sites in and adjacent to the CNS of a fourth PDGF-B lox/-organism, where the upper portion of the RE bar, marked with a triangle, indicates regions of increased pericyte density and the asterisk denotes sparse regions with densities similar to the rest of the CNS; Figure 18A shows the retina of a fifth PDGF-B lox/-organism; Figure 18B shows pericyte density and distribution in the retina of a fifth PDGF-B lox/-organism, where the asterisk indicates a region of sparse pericyte density and the triangle indicates a region of increased pericyte density; Figure 18C shows pericyte density and distribution in the pial vascular plexus of a fifth PDGF-B lox/-organism; Figure 18D shows pericyte density and distribution in the cerebellum of a fifth PDGF- B lox/-organism; Figure 18E depicts the relative quantification of pericyte density at four distinct sites in and adjacent to the CNS of a fifth PDGF-B lox/-organism, where the upper portion of the RE bar, marked with a triangle, indicates regions of increased pericyte density and the asterisk denotes sparse regiong with densities similar to the rest of the CNS; Figure 19 shows a portion of an adult wild type retina including a branched artery with its associated capillary network, where A indicates an artery and the arrow points to a pericyte; Figure 20 shows a portion of a retina from a PDGF-B lox/-mouse with very few pericytes and a large number of tortous and irregular vessels, where the arrows point to pericytes; Figure 21 shows a portion of a retina from a PDGF-B lox/-mouse with a chaotic, highly proliferative vasculature with highly increased numbers of endothelial cells and pericytes; Figure 22 shows an example of the capillary network in a PDGF-B lox/-mouse with more than 50% of the normal pericyte coverage, where the arrows indicate microaneurysms and the arrowhead indicates an abnormal ring structure; Figure 23 shows an example of the capillary network in a PDGF-B lox/-mouse with more than 50% of the normal pericyte coverage, where the arrows indicate regression profiles; Figure 24 shows a low magnification view of a region in a PDGF-B lox/-mouse with normal vascular density to the left bordering to a proliferative region to the right; Figure 25 shows a non-proliferative area of a PDGF-B lox/-mouse, where the arrowheads indicate regression profiles and the arrows indicate pyknotic nuclei; Figure 26 shows a high-magnification view of a proliferative region of a PDGF-B lox/-mouse; Figure 27A shows a z-scan through a control retina showing the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL) and outer nuclear layer (ONL); Figure 27B is taken from region 1 of Figure 27A and shows the levels of vascular plexus; Figure 27C is taken from region 2 of Figure 27A and shows the levels of vascular plexus; Figure 27D is taken from region 3 of Figure 27A and shows the levels of vascular plexus; Figure 28A shows a z-scan through a non-proliferative PDGF-B lox/-retina; Figure 28B is taken from region 1 of Figure 28A and shows the levels of vascular plexus; Figure 28C is taken from region 2 of Figure 28A and shows the levels of vascular plexus; Figure 28D is taken from region 3 of Figure 28A and shows the levels of vascular plexus; Figure 29A shows a z-scan through a non proliferative PDGF-B lox/-retina, where the arrow indicates a vessel penetrating from the choroid; Figure 29B is taken from region 1 of Figure 29A and shows the levels of vascular plexus; Figure 29C is taken from region 2 of Figure 29A and shows the levels of vascular plexus; Figure 29D is taken from region 3 of Figure 29A and shows the levels of vascular plexus, where the arrow indicates a vessel penetrating from the choroid; Figure 30A shows a z-scan through a PDGF-B lox/-retina, which is in the proliferative state, where the arrow indicates scattered microglial cells in the two deeper vascular plexus; Figure 30B is taken from region 1 of Figure 30A and shows the levels of vascular plexus in the proliferative state; Figure 30C is taken from region 2 of Figure 30A and shows the levels of vascular plexus where the vessels are regressing; Figure 30D is taken from region 3 of Figure 30A and shows the level of vascular plexus, where the arrow indicates scattered microglial cells and the vessels have regressed; Figure 31 shows a control eye with hematoxylin and eosin staining and X-gal positive pericytes; Figure 32A shows a PDGF-B lox/-eye with hematoxylin and eosin staining and X-gal positive pericytes, where the rectangles indicate the region magnified in Figures 32B and 32C; Figure 32B shows a magnified view of the region indicated by the upper rectangular box in Figure 32A, showing pericyte covered vessels in the vitreous; Figure 32C shows a magnified view of the region indicated by the lower (larger) rectangular box in Figure 32A, showing folding of the photoreceptor layer and disorganized neural layers in a severly affected retina; Figure 33 graphs pericyte density and proliferative retinopathy, where the highest pericyte density is set at 100% and the others are ranked relative thereto, and where all thirteen PDGF-B lox/-and PDGF-B lox/lox individuals with pericyte density less than 53% showed proliferative retinopathy affecting at least one eye; Figure 34 shows the outer retinal capillary plexus in a PDGF-B lox/-individual, where the arrowheads indicate regression profiles, the arrows indicate complex regression profiles typical for the PDGF-B lox/-individuals and P represents lacZ stained pericytes; Figure 35 graphically represents inter-individual inverse correlation between pericyte density in the cerebellum and the density of occlusions; Figure 36A shows a first region of a PDGF-B lox/-retina, with near-normal pericyte density and few regression profiles; Figure 36B shows a second region of the PDGF-B lox/-retina of Figure 36A, with moderate pericyte deficiency; Figure 36C shows a third region of the PDGF-B lox/-retina shown in Figures 36A and B, with near-complete pericyte deficiency correlating with a high number of regression profiles; Figures 37A-D represent quantative analysis that demonstrate the inverse correlation between pericyte count (Pc) and the number of occlusions (Occl) with a line connecting corresponding counts from the same microscopic field; Figure 37A shows data for a control individual; Figure 37B shows data for a first PDGF-B lox/-individual, including data from one area which had an almost completely regressed outer plexus and a low number of both pericytes and occlusions; Figure 37C shows data for a second PDGF-B lox/-individual; Figure 37D shows data for a third PDGF-B lox/-individual; Figure 38A shows a double-stained whole-mount preparation of a wild type thalamus, where the bar represents 100 am ; Figure 38B shows a double-stained whole-mount preparation of a wild type cerebellum, where the bar represents 100 u. m, pi indicates pial layer, and wm indicates white matter; Figure 39A shows a double-stained whole-mount preparation of a portion of a PDGF- B lox/-thalamus, analogous to that portion shown in Figure 38A, where the arrows indicate microaneurysms and the arrow-heads indicate normal-shaped vessels, and where the bar represents 100 um ; Figure 39B shows a double-stained whole-mount preparation of a portion of a PDGF- B lox/-cerebellum, analogous to that portion shown in Figure 38B, where the bar represents 100 am ; Figure 40A shows a PDGF-B lox/-mouse, where the inset arrow indicates the location of a micrograph; and Figure 40B shows a PDGF-B lox/-mouse with spontaneous cerebral bleeding and local reactive gliosis at the bleeding site, where the inset arrow indicates the location of bleeding and the location of the micrograph in the sample and ra represents reactive astrocytes.

DETAILED DESCRIPTION OF THE INVENTION In the present invention, diabetes was bypassed as an etiological agent for pericyte loss in mice. Endothelium-restricted ablation of PDGF-B led to a variable degree of pericyte loss in the CNS. Less than 50% pericyte loss promoted retinal changes typical of early, non- proliferative, diabetic retinopathy. More than 60% pericyte loss led to advanced, proliferative, retinopathy. Pericyte loss is a primary consequence of endothelial PDGF-B ablation. Data obtained from animal models according to the present invention suggest that pericyte loss is sufficient to cause retinopathy reminiscent of diabetic retinopathy. A threshold density of pericytes determines if progression from non-proliferative to proliferative retinopathy will occur.

The Cre-loxP (causes recombination-locus of crossing over) recombination system was used to target PDGF-B synthesis in endothelial cells in mice. This system was isolated from bacteriophage PI and consists of two 13 bp protein binding regions separated by an 8 bp spacer region, which is recognized by Cre recombinase, a 35 kDa protein. Nucleic acid sequences for loxP and Cre are known. The Cre protein catalyzes a site-specific recombination event. This event is bidirectional, i. e., Cre will catalyze the insertion of sequences at a loxP site or excise sequences that lie between two loxP sites. Thus, if a construct for insertion also has flanking loxP sites, introduction of the Cre protein, or a polynucleotide encoding the Cre protein, into the cell will catalyze the removal of the construct DNA. Further descriptions of this technology can be found in, inter alia, U. S.

Patent No. 4,959, 317, the disclosure of which is incorporated herein by reference.

The PDGF-B synthesis targeting resulted in postnatally viable mutants in which a portion of the endothelial cells failed to express PDGF-B. Endothelium-derived PDGF-B has now been found to play a critical role in the recruitment of pericytes to the vessels penetrating into the embryonic CNS. Through breakthroughs made possible by the present invention, endothelium-restricted PDGF-B deficiency has been shown to lead to pericyte loss that persists in the adult and leads to retinal and brain microvasculature abnormalities with characteristics of diabetic microangiopathy. Surprisingly, there is substantial inter-individual variability in both the extent of PDGF-B loss in the capillary endothelium and in the extent of pericyte loss.

Previous studies have shown that endothelial cells are one of several sources of PDGF-B in the developing mouse embryo, and that pericytes expressing PDGFR depend on PDGF-B/PDGFRß signaling for their recruitment to newly formed vessels. By generating endothelium-restricted PDGF-B knockout mice, endothelium-derived PDGF-B was established as a promoter of pericyte recruitment to CNS vessels during developmental angiogenesis. The Cre-mediated recombination frequency in endothelial cells and the consequent reduction in pericyte coverage of CNS microvessels varied between animals of the same genotype, generating a spectrum of individuals with endothelial PDGF-B ablation and pericyte loss ranging between 40-90%.

In animals with up to 50% pericyte loss (or, those displaying more than 50% of normal overall CNS pericyte density), the retinal vasculature showed changes similar to those seen in early retinopathy in humans and in diabetic rodents, such as an increased density of microvessels with irregular diameter, signs of vascular regression and vascular sprouting, and occasional microaneurysms and tortuous microvessels. In animals with more than 50% pericyte loss (or, less than 50% normal pericyte density) the retinas developed many of the hallmarlcs of proliferative retinopathy. For example, there was a massive increase in the number of vessels and an overall increase in both endothelial cells and pericytes. Highly abnormal vessels penetrated into the vitreous and lens, and also through the retina to fuse with choroidal vessels. Since pericyte loss is the primary event in the endothelium-restricted PDGF-B knockout model, data obtained through the present invention establish a causal link between pericyte loss and microangiopathy, including non-proliferative as well as proliferative retinopathy.

The pericyte proliferation associated with these proliferative changes is apparently independent of endothelium-derived PDGF-B, showing different mechanisms govern pericyte proliferation in association with normal or pathological angiogenesis. Regions of vascular proliferation in the inner plexus, complete outer plexus regression, and vitreous-and choroid neoangiogenesis, correlated without exception, and bordered sharply to non- proliferative regions. The inverse correlation between the numbers of pericytes and regressing capillaries at both the inter-and intra-individual levels strongly suggests that pericyte deficiency triggers capillary occlusion. This may be tolerable up to a threshold level, above which neoangiogenic responses are initiated leading to proliferative retinopathy.

Data obtained from experimental animals of the present invention show the triggering of pericyte loss, independent of diabetes, leads to a wide spectrum of changes characteristic of diabetic retinopathy. This data shows pericyte loss constitutes a critical causal linlc in the chain of pathogenic events leading to diabetic retinopathy. This data also shows that a threshold level of pericyte loss determines if progression to proliferative retinopathy will occur.

Further data obtained from novel animals according to the present invention suggest that retinal vessels are more sensitive to pericyte loss than vessels elsewhere in the CNS. Up to 50% pericyte loss did not promote any characteristic signs of microangiopathy in the frontal cortex, thalamus or cerebellum. In contrast, 80-90% pericyte loss led to changes in brain microvessels similar to those apparent in the retina at 50% pericyte loss, i. e. tortuous capillaries with irregular diameter and occasional microaneurysms. Focal signs of astroglial reaction throughout the CNS implicated focally dysfunctional vessels. Together with the high pericyte to endothelial ratio seen normally in the retina, this data implies a particularly important role for pericytes in the retinal microvasculature. This explains why the retinal vasculature appears particularly sensitive to hyperglycemia. Based on this new data, researchers in many fields can develop new approaches to testing, treating, and potentially preventing undesirable outcomes such as vision impairment in diabetic humans.

Materials and equipment used in the experiments described below are commercially available. In some instances, specific sources are given, however, sources or instructions to make the materials are known to skilled workers. While the detailed experiments involve murine species, it is within the scope of the present invention to utilize various other non- human mammals, such as rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs and monkeys.

The applicability and transferability of the present invention to other species relies in part on the evolutionary conservation of cell signaling pathways. Further, the present invention utilized a Cre-loxP recombination system to delete exon 4 of PDGF-B and create a research model organism. Other means to delete exon 4, and alternate approaches to inactivate PDGF- B are known in the art, for example deletion of the entire PDGF-B gene. Whichever method is most convenient and reliable or otherwise favored by a researcher could be employed to practice the present invention.

Example 1: Generation of Mice with Endothelium-Restricted Deletion of PDGF-B Deletion of exon 4 in the gene encoding the PDGF-B protein is known to result in a null allele5. The location of this exon in the human genome is described in Dalla-Favera et al., Chromosomal localization of the human homo log (c-sis) of the simian sarcoma virus onc gene, Science 12 ; 218 (4573): 686-8 (November 1982) and Swan et al., Chromosomal mapping of the simian sarcoma virus onc gene analogue in human cells, Proc Natl Acad Sci U S A 79 (15): 4691-5 (August 1982). Further references describing this exon in various species are known in the art. LoxP sites were positioned on each side of exon 4 by homologous recombination in ES-cells, using a two-step procedure outlined in Figure 1.

Specifically, a targeting vector was generated in which exon 4, which codes for the major part of the PDGF-B protein, was surrounded by a loxP-flanked PGK-neo cassette (neo refers to the neomycin resistance gene) in intron 3 and a single loxP fragment in intron 4.

About 9 kb of homology, including exons 2 and 3, was added upstream from the loxP-neo cassette and about 3.5 lcb of 3'homology, including exon 5, was added downstream from the single loxP sequence (Fig. 1). Cre-mediated recombination between the loxP sites in the construct was confirmed in Cre expressing bacteria.

The targeting vector was linearized with NotI and transfected by electroporation into E14. 1 ES cells, which were G418 selected and processed as described by Leveen, et al. 5 Southern blotting identified homologous recombination at a very high frequency (one fifth of the G418 resistant clones). A high proportion (more than 50%) of the recombinant ES-clones had a concatamer of two or more copies of the targeting construct integrated at the PDGF-B locus. Those ES-clones in which a single copy of the targeting construct had integrated correctly at the PDGF-B locus were identified. Of these clones, about one-third contained the loxP site in intron 4 and two thirds were lacking it, showing that recombination could take place on either side of the loxP site.

One ES-clone containing a single correctly integrated targeting construct (flox-neo allele) was chosen for deletion of the loxP-flanlced PGK-neo cassette. Approximately 5X106 ES-cells were transiently transfected with l0ug of hCMV-Cre expression plasmid pBS185 (Philippe Soriano, Seattle, USA) seeded at clonal density and allowed to form colonies at standard ES-cell culture conditions for 10 days. Clones in which the PGK-neo cassette was deleted but PDGF-B exon 4 remained (flox allele, that is,"floxed"or flanked on either side by a lox-P site) were distinguished from clones in which recombination had deleted both PGK-neo and exon 4 (lox allele) by southern blot analysis of G418 sensitive clones. ES-cell clones carrying the flox allele by were injected into C57BL/6 blastocystsS to create chimeric, heterozygous (PDGF-B flox/+) and homozygous (PDGF-B flox/flox) mice.

Of three chimeric males generated, one gave offspring carrying the PDGF-B flox allele. Intercrossing of PDGF-B flox/+ mice gave rise to PDGF-B flox/flox homozygotes at Mendelian frequency. PDGF-B flox/-mice were generated by intercrossing PDGF-B flox carriers with PDGFB +/-mice5. PDGF-B flox/-mice were born at the expected Mendelian frequency, reached adulthood and were phenotypically indistinguishable from PDGF-B+/- mice. This and other analysis indicated that the PDGF-B flox allele was functionally equivalent with the PDGF-B wild type allele.

A three-primer PCR protocol was established for discrimination between the wild type, flox and lox alleles as shown in Figure 1. PDGF-B flox/flox mice were normal, demonstrating that the flox allele retained functional activity. PDGF-B flox/-animals were generated by intercrossing PDGF-B +/-mice5. These mice were also found to be normal, in agreement with the lack of a discernable phenotype in PDGF-B +/-mice.

Mice expressing the Cre recombinase from the Tie-1 promoter, TielCres l3 were crossed with PDGF-B+/-mice and, subsequently, with PDGF-B flox/flox mice to generate endothelium-restricted PDGF-B gene inactivation. In the litters of such crosses, TielCre+PDGF-Bflox/- ("lox/-") constitutes the endothelium-restricted knockouts, whereas TielCre°PDGF-Bflox/+ ("flox/+"), TielCre°PDGF-Bflox/- ("flox/-") and TielCre+PDGF- Bflox/+ ("lox/+") represent various types of controls. In contract to PDGF-B-/-mice ("-/-"), which invariably die perinatally5, lox/-mice were born at Mendelian frequency, reached adulthood and were fertile.

To assess the efficiency of Cre-mediated recombination at the PDGF-B locus in endothelial cells in vivo, capillary fragments consisting of 80% endothelial and 20% non- endothelial cells were isolated from brains of embryonic day 16.5 (E16. 5) lox/+ mouse embryos, and the relative abundance of the flox and lox alleles determined by PCR (Fig. 4).

The intensity of the wild type allele PCR fragment was similar in all individuals, serving as a control for the PCR reaction. The different lanes (1-9) of Figure 4 represent different animals. Although they all had the same genotype (lox/+), assuming equal efficiency of amplification of the wild type, flox and lox alleles, the Cre-mediated recombination of the flox allele in endothelial cells from different individuals varied between approximately 20- 90%.

The variation ranged from approximately 20% in individual number 10 to more than 70% in individuals number 2 and 3. The capillary fragments contain around 20% non- endothelial cells, comprised of approximately 10% pericytes and 10% non-capillary cells (data not shown). This shows that the recombination frequency in E16.5 brain endothelial cells ranges from 40% to 90% or more. In postnatal litters, lox/-mice appeared with Mendelian distribution (data not shown), reached adulthood and were fertile. This contrasts to PDGF-B-/-mice, which invariably die perinatally5.

Example 2: Endothelium-specific PDGF-B deletion leads to impaired pericyte recruitment to brain microvessels Tie-1 Cre transgenic mice13 that express Cre under the mouse tie-1 promoterl8 were mated with PDGF-B +/-mice to generate Tiel+Cre PDGF-B+/-offspring. These offspring were subsequently crossed with PDGF-Bflox/flox mice to create Tiel Cre PDGF-B lox/- (lox/-) animals as well as various controls (Tiel+Cre+, flox/+, Tiel Cre, flox/-, TielCre°, lox/+). The mice were bred onto the background of the XlacZ4 reporter mice (Pericyte-LacZ) that express-galactosidase in vSMC/PC14. The XlacZ4 transgenic mouse, in which lacZ expression is restricted to vascular smooth muscle cells and pericytes from late gestation onwards in correlation with other vSMC and pericyte markers, such as alpha- smooth muscle actin and desmin, was used to facilitate visualization and quantification of pericyte recruitment to brain microvessels. In some of the subsequent crosses, lox/-mice were intercrossed, which generated, in addition to the above noted genotypes, TielCre+, lox/lox individuals, in which Cre must inactivate two alleles in each cell to render a null situation, and PDGF-B-/-individuals (embryonic lethal).

PCR was used to genotype the mice, using the primers: BF-5'-GGGTGGGACTTTGGTGTAGAGAAG-3' (SEQ ID NO : 1), BB1-5'-TTTGAAGCGTGCAGAATGCC-3' (SEQ ID NO : 2), BB2-5'-GGAACGGATTTTGGAGGTAGTGTC-3' (SEQ ID NO : 3), and BBlox-5'-TCTGGGTCACTGCTTCAGAATAGC-3' (SEQ ID NO : 4).

For genotyping mice during breeding, a 3-primer PCR genotyping protocol using the BF (a common forward primer), BB2 (a reverse primer in the neo cassette) or BBlox (a reverse primer in intron 4) primer combined with tail or toe DNA in Gittchier buffer. Forty cycles of PCR were run as follows: 96°C for 30 seconds, 57. 9°C for 30 seconds, 65°C for 2 minutes.

This protocol generates diagnostic fragments of 265,624, 400 and 540 bp for the wild-type (PDGFB+), PDGF-B null alleles (PDGFB-), flox (PDGFB-), and lox (PDGFB-), respectively (Fig 3).

PCR genotyping of the mice also enabled observation of the extent of recombination in vivo. Microvascular fragments were isolated from brains of E16. 5 lox/+ embryos and were prepared according to the method described in Gargett et al. 30. The isolated fragments contained about 80% endothelial cells, 10% pericytes and 10% non-vascular cells as judged by marker analysis. DNA isolated from the microvascular fragments was subjected to the PCR genotyping protocol described above.

Pericyte abundance and distribution were determined by whole mount staining of E15. 5 brains. Figures 5-8 show the pericyte distribution in the perineural vascular plexus of wild type, PDGFB-/-and lox/-individuals. Beta-galactosidase of the XlacZ4 transgene product staining was used reveal pericyte nuclei (blue). Two lox/-individuals with different degree of pericyte loss are shown, while one of the two lox/-individuals show a pericyte reduction comparable in magnitude to the-/-embryo, the other shows a pericyte abundance intermediate between those of-/-and wild type embryos.

The pericyte distribution was further analyzed in the developing midbrain (Figures SB, 6B, 7B, 8B). Vascularization of this region follows a stereotyped pattern as blood vessels enter the brain tissue perpendicular to the surface (radial branches) and ramify and form a capillary network in the subventricular zone. Pericytes were found in association with the radial branches as well as with the subventricular plexus. Only very few pericytes were found at any of these locations in the-/-tissue. The lox/-phenotype was intermediate between wild type and-/-, and was variable between different lox/-individuals.

Local bleedings and dilated capillaries were seen in-/-as well as in lox/-individuals but not in controls. A close inspection of the lox/-radial branches showed that although most of them lacked or had reduced numbers of pericytes, occasional branches had normal pericyte coverage. This distribution would be expected from a mosaic situation with regard to endothelial PDGF-B expression and is in agreement with the genetic data regarding efficiency of Cre-mediated recombination in endothelial cells.

Example 3: Persistent pericyte reduction in the CNS leads to microangiopathy and proliferative retinopathy XlacZ4 staining of brains from 3-week-old mice of different genotypes according to Example 2, above, revealed that the pericyte deficiency of lox/-mice did not normalize postnatally. XlacZ4 positive pericytes and vascular smooth muscle cells were reduced in number in the lox/-mice in comparison with controls at all sites investigated in the CNS and its pial vessels (some data in Figures 9-12, some data not shown). This reduction affected arteries, veins and capillaries. As a consequence, long stretches of vessels of all types lacked associated XlacZ4 positive cells (Fig. 12B, arrowheads show examples of forebrain pial arterial branches). The persistence of pericyte deficiency in postnatal lox/-mice can be seen in Figures 9 and 11, where brains of 21-day-old wild-type and lox/-mice were whole-mount stained for the XlacZ4 pericyte marker. There was only a sparse coat of XlacZ4 positive cells on the lox/-vessels.

Quantification of pericyte abundance in four different regions of the brain (pial plexus, forebrain cortex, thalamus and cerebellum) showed that the level of reduction was constant between different regions in the same individual, but varied extensively between individuals (Figures 13-18). The level of reduction of pericytes in the retina mimicked that of the rest of the CNS when pericyte loss was less than 50% (Figures 13-16). When pericyte loss exceeded 50% at other CNS sites, the retinal vasculature was progressively deranged.

This correlated with retinal traction, in-growth of pigment epithelial cells, and increased number of XlacZ4+ cells (Figures 16-18).

Figures 13A through 18E illustrate the correlations between pericyte loss in the CNS and development of advanced retinopathy. The Figures compare six individuals; one wild- type and five different lox/-individuals. In the Figure'E'corresponding to each number 13- 18, the densities are shown relative to the wild type (set to 100%). Individuals represented in Figures 14 and 15 have less than 50% pericyte loss at other sites in the CNS yet show a similar degree of pericyte loss in the retina. Individuals represented in Figures 16,17 and 18 have more than 60% pericyte loss at most sites investigated and show advanced changes to the retina with severe vessel abnormalities and retinal traction. In the retinas for Figures 16- 18, it can be seen that the density of XlacZ4 positive pericytes is largely increased.

While the overall retinal vascular pattern was normal in mice with less than 50% reduction of CNS pericytes, the morphology of the retinal capillary network was abnormal including an increased density of capillary branches with variable capillary diameter and occasional microaneurysms (Figures 19-26). Double staining of whole mount retinas for endothelial cells (fluorescent green) and pericytes (blue/black, color not depicted in figures) helps illustrate retinopathy in lox/-mice. While the individual represented in Figure 19 showed increased vessel density with tortuous and irregular vessels, the individuals represented in Figures 20 and 21 show a highly proliferative vasculature with highly increased numbers of endothelial cells as well as pericytes. The new vessels in Figures 20 and 21 are highly irregular in shape and diameter.

Retinas in mice with more than 60% reduction of CNS pericytes showed complete loss of normal vascular pattern and the presence of highly abnormal vascular structures with increased numbers of endothelial cells as well as pericytes. Vascular structures penetrated the retina to fuse with choroidal vessels and also invaded the vitreous and lens. A reduction of the pericyte density in the CNS by more than 60% correlated with the development of proliferative retinopathy.

Microvessel proliferation was not seen outside the retina, but there were clear signs of microangiopathy throughout the CNS in animals with 80-90% pericyte loss. For example, forebrain cortex, thalamus and cerebellum all showed presence of tortuous irregular diameter capillaries with multiple microaneurysms (Figures 38 and 39). In cerebellum a significant reduction in microvessel density was also apparent. Multiple foci of reactive astrocytes were found throughout the CNS, often associated with visible microaneurysms. The microvascular abnormalities caused by pericyte loss promotes astroglial responses typical of multifocal CNS injury.

For Figures 38 and 39, double staining of endothelium (brown) and pericytes (blue, colors not depicted in figures) in whole mount preparations of regions of the CNS depicted the mocriangiopathy in the CNS of lox/-mice. The lox/-individual represented in Figures 39A and 39B shows around 90% pericyte loss and had irregular vasculature and with tortuous vessels of uneven diameter, at decreased density, and the presence of microaneurysms. There is a direct correlation between the shape of individual vessels and pericyte density as vessels in the lox/-individuals with some pericytes retained show a relatively uniform shape and diameter.

Example 4: Histological analysis Whole mount beta-galactosidase stainings of whole tissue and sections were done as described by Hogan, et all9. Pericyte densities were quantified by counting XlacZ4 positive nuclei on images captured from whole mount preparations of various parts of the CNS (including the retina) or 200 pm thick vibratome sections studied in a dissection microscope at fixed magnification. First, an image area containing more than 300 lacZ positive nuclei in a wild type individual was located. Then, that area was counted in different lox/-individuals.

The number of pericytes in lox/-individuals is described as a percentage of the number of pericytes expected in a corresponding region of a wild type tissue.

X-gal stained retinas were post-fixed for 10 minutes in 4% paraformaldehyde, followed by isolectin staining (Bandeiraea simplicifolia, Sigma L-2140). Retinas were incubated in 1% BSA, 0.5% Tween in PBS overnight, washed twice in PBS pH 6.8 containing 1% Tween, O. 1mM CaC12, O. lmM MgCl2, O. lmM MnCl2 (PBlec) and incubated in biotinylated isolectin (20 llg/ml in PBlec) at 4°C overnight. Following washes in PBS, isolectin was detected using 101lg/ml of a fluorescent streptavidin conjugate (Alexa Fluor 488, Molecular Probes, S-11223). TO-PRO 3 (1: 1000; Molecular Probes) was used for nuclear counterstaining. Whole mount isolectin staining on post-fixed X-gal stained P21 brains was achieved using peroxidase-conjugated isolectin B4 from Bandeiraea simplicifolia (Sigma L-5391). Endogenous peroxidase was blocked prior to lectin staining using 0.6% H202 in PBS. Peroxidase activity was detected by standard DAB staining. Vibratome brain sections (200pm) were stained with isolectin B4 as described for retinas.

Endogenous peroxidase was blocked prior to lectin staining, by 0.6% H202 in PBS.

Peroxidase activity was detected by standard DAB staining. GFAP labeling was achieved using a polyclonal rabbit antibody (1: 75, Dako Z 0334) followed by Alexa-568 conjugated secondary antibody (Molecular Probes). Confocal images were taken on a Leica TCS NT microscope system and processed in Adobe Photoshop. Retinal vascular preparations were obtained using a pepsin-trypsin digestion technique. The combined pepsin- (5% pepsin in 0.2% hydrochloric acid for 120 minutes) and trypsin- (2. 5% in 0.2 M Tris for 15 to 30 minutes) digestion was used to isolate the retinal vasculature. Subsequently, the samples were stained with periodic acid Schiff's (PAS) stain, the method being further described by Hammes et al38.

Example 5: Capillary Regression in the Outer Plexus leads to Proliferative Retinopathy Two interconnected vascular plexus typically develop in the retina, an inner one with arteries, veins and capillaries, and an outer plexus built mainly of capillaries. In lox/- mutants, both plexus displayed characteristic abnormalities including vascular occlusion and regression. Simultaneously scoring of the frequency and pattern of capillary occlusion and pericyte loss was conducted in a number of lox/-mutants to address if this correlated directly with pericyte loss. Focusing on the outer vascular plexus, pericytes and occluded capillary branches were counted per microscopic field (Figs 37A-D).

Ten random fields were analyzed for each retina, representing together about half of the retinal area. Branch occlusions were observed in regular patterns in controls. This is known to be part of the normal vessel remodeling and it affects only singular branches interconnecting neighboring plexus units, as defined by supplying arterioles. In contrast, lox/-mutants displayed both an increased density of regression profiles and a distinctive change in their pattern. Several sequential branches were often affected, leading to the formation of characteristic Y-shaped, or more complex, regression profiles (Figs 36A-C).

In addition to an inter-individual correlation between pericyte loss and regression, there was a similar intra-individual correlation in different retinal regions (Fig 33). An inverse correlation between number of pericytes and number of regression profiles was seen in the different microscopic fields (Figs 36A-D). Complete loss of pericytes correlated with the complete regression of the outer plexus. This, in turn, correlated with the occurrence of proliferative changes at the retinal surface and the formation of abnormal vascular penetration of the bottom layers of photoreceptor and RPE cells, as shown by confocal z-scans (Figs 27A-30D).

Example 6: Microangiopathy outside the retina Lox/-mutants with more than 50% of the normal pericyte density lacked obvious signs of vascular abnormalities in parts of the CNS other than the retina. In animals with the lowest pericyte density, however, there were clear signs of microangiopathy throughout the CNS. In these individuals the forebrain cortex, thalamus and cerebellum all presented tortuous irregular diameter capillaries and multiple microaneurysms (Fig 39A and data not shown). A significant reduction of microvessel density was also apparent in both the thalamus and cerebellum of such mice (Figs 39A, 39B). These regions also displayed foci of reactive astrocytes and increased number of microglia in association with obvious vessel abnormalities such as microhemorrhage. Thus, the microvascular abnormalities caused in the CNS by severe pericyte loss promotes astroglial responses typical of small focal CNS injuries. A vascular proliferative response to pericyte loss, resulting in increased vessel density, was never seen in lox/-CNS outside the retina.

Example 7: Generation of Augmented Research Model Organism The present invention provides a research organism having an inactivated PDGF-B gene. This research model organism may be used to, inter alia, screen potential therapeutic compounds in the prevention, diminution or reversal of disease such as diabetic retinopathy.

When screening potential therapeutic compounds, it may facilitate testing to accelerate even further the retinopathic symptoms in the research organism.

One means for accelerating or providing additional retinopathic symptoms in the research organism would be to administer a diabetes inducing agent such as streptozotocinl227. Another way to accomplish the acceleration is to breed the research organism with a diabetic genetic model organism. For murine research organisms, the research organism could be bred with, for example, ob/ob mice. The ob/ob mice have a mutation in the obesity (ob) gene situated in the 7q31 chromosomal region. The peptide hormone encoded by this gene, leptin, is a plasma protein of 16 kDa produced by the adipocytes under the action of various stimuli and is involved in the mechanisms of satiety.

In ob/ob mouse plamsa, leptin is not detectable. The resultant strain would have the beneficial properties of the research organism described herein, and may have an accelerated or more aggressive course of disease with which to research and evaluate therapeutic treatments.

The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof. References relied upon in the disclosure are hereby incorporated by reference in their entirety.

REFERENCES 1. Sims, D. E. The pericyte-A review. Tissue and Cell 18, 153-174 (1986).

2. Betsholtz, C. , Karlsson, L. & Lindahl, P. Developmental roles of platelet-derived growth factors. BioEssays 23,494-507 (2001).

3. Benjamin, L. E. , Hemo, I. & Keshet, E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 125,1591-8 (1998).

4. Benjamin, L. E. , Golijanin, D. , Itin, A. , Pode, D. & Keshet, E. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal [see comments]. JClin Invest 103,159-65 (1999).

5. Levéen, P. et al. Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev. 8,1875-1887 (1994).

6. Soriano, P. Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev. 8,1888-1896 (1994).

7. Lindahl, P., Johansson, B. R., Levéen, P. & Betsholtz, C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277,242-5 (1997).

8. Hellström, M. , Kalen, M. , Lindahl, P. , Abramsson, A. & Betsholtz, C. Role of PDGF- B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 126, 3047-55 (1999).

9. Hellström, M. et al. Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. JCell Biol 153, 543-553 (2001).

10. Cogan, D. G. , Toussaint, D. & Kuwabara, T. Retinal vascular patterns. IV. Diabetic retinopathy. Archs. Opthalmol. 66,366-378 (1961).

11. Kuwabara, T. & Cogan, D. G. Retinal vascular patterns. VI. Mural cells of the retinal capillaries. Archs Ophtal 69,492-502 (1963).

12. Engerman, R. L. Pathogenesis of diabetic retinopathy. Diabetes 38,1203-1206 (1989).

13. Gustafsson, E., Brakebusch, C. , Hietanen, K. & Fassler, R. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice. J Cell Sci 114, 671-6. (2001).

14. Tidhar, A. et al. A novel transgenic marlcer for migrating limb muscle precursors and for vascular smooth muscle cells. Dev D 220,60-73. (2001).

15. Klinghoffer, R. A. , Mueting-Nelsen, P. F., Faerman, A. , Shani, M. & Soriano, P. The two PDGF receptors maintain conserved signaling in vivo despite divergent embryological functions. Mol. Cell 7, 343-354 (2001).

16. Abramsson, A. et al. Analysis of mural cell recruitment to tumor vessels. Circulation 105,112-117 (2002).

17. Aiello, L. P. et al. Diabetic retinopathy. Diabetes Care 21,143-156 (1998).

18. Korhonen, J. et al. Endothelial-specific gene expression directed by the tie gene promoter in vivo. Blood 86,1828-35. (1995).

19. Hogan, B. , Beddington, R. , Costantini, F. & Lacy, E. Manipulating the mouse embryo; a laboratory manual, (Cold Spring Harbor Laboratory Press, 1994).

20. Antonetti, D. A. , Lieth, E. , Barber, A. J, & Gardner, T. W. Molecular mechanisms of vascular permeability in diabetic retinopathy. Semin, Ophtalmol. 14,240-248 (1999).

21. Aiello, L. M. , Cavallerano, J. D. and Aiello, L. P. Diagnosis, management, and treatment of non-proliferative diabetic retinopathy and macular edema. Principles and Practice of Ophthalmology, D. M. Albert and F. A. Jacobiec, eds. (Philadelphia, W. B.

Saunders Company), pp 1900-1914 (2000).

22. Thylefors, B. , Negrel, A. D. , Pararajasegaram, R. & Dadzie, K. Y. Global data on blindness. Bull. World Wealth Organ 73, 115-121 (1995).

23. Miller, J. W. and D'Amico, D. J. Proliferative diabetic retinopathy. Principles and Practice of Ophthalmology, D. M. Albert and F. A. Jacobiec, eds. (Philadelphia, W. B.

Saunders Company), pp 1915-1945 (2000).

24. Brownlee, M. Biochemistry and molecular cell biology of diabetic complications.

Nature 41,813-820 (2001).

25. Lieth, E. et al. Glial reactivity and impaired glutamate metabolism in short-term experimental diabetic retinopathy. Diabetes 47,815-820 (1998).

26. Buzney, S. M., Frank, R. N. , Varma, S. D. , Tanishima, T. & Gabbay, K. H. Aldose reductase in retinal mural cells. Invest. Ophtal. Vis Sci. 16,392-396 (1997).

27. Buscher, C. et al. Islet transplantation in experimental diabetes of the rat. XII. Effect on diabetic retinopathy. Morphological findings and morphometrical evaluation.

Horion. Metab. Res., 21,227-231 (1989).

28. Klein, R., Klein, B. , Moss, S., et al. Diabetic retinopathy. II : Prevalence and risk of diabetic retinopathy when age at diagnosis is less than 30 years. Arch Opthalmol 105, 520-526 (1984).

29. Klein, R. , Klein, B. , Moss, S., et al. The Wisconsin epidemiologic study of diabetic retinopathy. III : Prevalence and risk of diabetic retinopathy when age at diagnosis is 30 or more years. Arch Opthalmol, 105,527-532 (1984).

30. Gargett, C. E., Bucak, K. & Rogers, P. A. Isolation, characterization and long-term culture of human myometrial microvascular endothelial cells. Hum. Reprod. 15,293- 301 (2000).

31. Suri, C. et al. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic development. Cell 87,1171-1180 (1996).

32. Patan, S. TIE1 and TIE2 receptor tyrosine kinases inversely regulate embryonic angiogenesis by the mechanism of intussusceptive microvascular growth. Microvasc Res 56,1-21 (1998).

33. Li, D. Y. et al. Defective angiogenesis in mice lacking endoglin. Science 284,1534-7 (1999).

34. Yang, X. et al. Angiogenesis defects and mesenchymal apoptosis in mice lacking SMAD5. Development 126,1571-80 (1999).

35. Oh, S. P. et al. Activin receptor-like kinase 1 modulates transforming growth factor- beta 1 signaling in the regulation of angiogenesis. Proc Natl Acad Sci USA 97,2626- 31 (2000).

36. Liu, Y. et al. Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J. Clin. Invest. 106,951-961 (2000).

37. Stalmans, 1. et al. Arteriolar and venolar patterning in retinas of mice selectively expressing VEGF isoforms. J Clin. Invest. 109,327-336 (2002).

38. Hammes, H. -P., Martin, S. , Federlin, K. , Geisen, K. & Brownlee, M. Aminoguanidine treatment inhibits the development of experimental diabetic retinopathy. Proc. Natl.

Acad. Sci. USA 88, 11555-11558 (1991).