Chemical interrogation of FOXO3a nuclear translocation identifies potent and selective inhibitors of phosphoinositide 3-kinases.

Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is one the most frequent genetic events in human cancer. A cell-based imaging assay that monitored the translocation of the Akt effector protein, Forkhead box O (FOXO), from the cytoplasm to the nucleus was employed to screen a collection of 33,992 small molecules. The positive compounds were used to screen kinases known to be involved in FOXO translocation. Pyrazolopyrimidine derivatives were found to be potent FOXO relocators as well as biochemical inhibitors of PI3Kalpha. A combination of virtual screening and molecular modeling led to the development of a structure-activity relationship, which indicated the preferred substituents on the pyrazolopyrimidine scaffold. This leads to the synthesis of ETP-45658, which is a potent and selective inhibitor of phosphoinositide 3-kinases and demonstrates mechanism of action in tumor cell lines and in vivo in treated mice.

Genetic modelling of the PTEN/AKT pathway in cancer research.

The focus on targeted therapies has been fuelled by extensive research on molecular pathways and their role in tumorigenesis. Novel models of human cancer have been created to evaluate the role of specific genes in the different stages of cancer. Currently, mouse modelling of human cancer is possible through the expression of oncogenes, specific genetic mutations or the inactivation of tumour suppressor genes, and these models have begun to provide us with an understanding of the molecular pathways involved in tumour initiation and progression at the physiological level. Additionally, these mouse models serve as an excellent system to evaluate the efficacy of currently developed molecular targeted therapies and identify new potential targets for future therapies. The PTEN/AKT pathway is implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. Deregulation of the PTEN/AKT pathway is a common event in human cancer. Despite the abundant literature, the physiological role of each element of the pathway has begun to be uncovered thanks to genetically engineered mice. This review will summarise some of the key animal models which have helped us to understand this signalling network and its contribution to tumorigenesis.

Cooperation between Cdk4 and p27kip1 in tumor development: a preclinical model to evaluate cell cycle inhibitors with therapeutic activity.

Deregulation of the G1-S transition of the cell cycle is a common feature of human cancer. Tumor-associated alterations in this process frequently affect cyclin-dependent kinases (Cdk), their regulators (cyclins, INK4 inhibitors, or p27Kip1), and their substrates (retinoblastoma protein). Although these proteins are generally thought to act in a linear pathway, mutations in different components frequently cooperate in tumor development. Using gene-targeted mouse models, we report in this article that Cdk4 resistance to INK4 inhibitors, due to the Cdk4 R24C mutation, strongly cooperates with p27(Kip1) deficiency in tumor development. No such cooperation is observed between Cdk4 R24C and p18(INK4c) absence, suggesting that the only function of p18INK4c is inhibiting Cdk4 in this model. Cdk4(R/R) knock in mice, which express the Cdk4 R24C mutant protein, develop pituitary tumors with complete penetrance and short latency in a p27Kip1-/- or p27Kip1+/- background. We have investigated whether this tumor model could be useful to assess the therapeutic activity of cell cycle inhibitors. We show here that exposure to flavopiridol, a wide-spectrum Cdk inhibitor, significantly delays tumor progression and leads to tumor-free survival in a significant percentage of treated mice. These data suggest that genetically engineered tumor models involving key cell cycle regulators are a valuable tool to evaluate drugs with potential therapeutic benefit in human cancer.

Absence of glial fibrillary acidic protein and vimentin prevents hypertrophy of astrocytic processes and improves post-traumatic regeneration.

The regenerative capacity of the CNS is extremely limited. The reason for this is unclear, but glial cell involvement has been suspected, and oligodendrocytes have been implicated as inhibitors of neuroregeneration (Chen et al., 2000, GrandPre et al., 2000; Fournier et al., 2001). The role of astrocytes in this process was proposed but remains incompletely understood (Silver and Miller, 2004). Astrocyte activation (reactive gliosis) accompanies neurotrauma, stroke, neurodegenerative diseases, or tumors. Two prominent hallmarks of reactive gliosis are hypertrophy of astrocytic processes and upregulation of intermediate filaments. Using the entorhinal cortex lesion model in mice, we found that reactive astrocytes devoid of the intermediate filament proteins glial fibrillary acidic protein and vimentin (GFAP-/-Vim-/-), and consequently lacking intermediate filaments (Colucci-Guyon et al., 1994; Pekny et al., 1995; Eliasson et al., 1999), showed only a limited hypertrophy of cell processes. Instead, many processes were shorter and not straight, albeit the volume of neuropil reached by a single astrocyte was the same as in wild-type mice. This was accompanied by remarkable synaptic regeneration in the hippocampus. On a molecular level, GFAP-/-Vim-/- reactive astrocytes could not upregulate endothelin B receptors, suggesting that the upregulation is intermediate filament dependent. These findings show a novel role for intermediate filaments in astrocytes and implicate reactive astrocytes as potent inhibitors of neuroregeneration.

Pericytes and the pathogenesis of diabetic retinopathy.

Pericytes provide vascular stability and control endothelial proliferation. Pericyte loss, microaneurysms, and acellular capillaries are characteristic for the diabetic retina. Platelet-derived growth factor (PDGF)-B is involved in pericyte recruitment, and brain capillaries of mice with a genetic ablation of PDGF-B show pericyte loss and microaneurysms. We investigated the role of capillary coverage with pericytes in early diabetic retinopathy and the contribution to proliferative retinopathy using mice with a single functional allele of PDGF-B (PDGF-B(+/-) mice). As assessed by quantitative morphometry of retinal digest preparations, pericyte numbers in nondiabetic PDGF-B(+/-) mice were reduced by 30% compared with wild-type mice, together with a small but significant increase in acellular capillaries. Pericyte numbers were reduced by 40% in diabetic wild-type mice compared with nondiabetic wild-type controls. Pericyte numbers were decreased by 50% in diabetic PDGF-B(+/-) mice compared with nondiabetic wild-type littermates, and the incidence of acellular capillaries was increased 3.5-fold when compared with nondiabetic PDGF-B(+/-) mice. To investigate the effect of pericyte loss in the context of ongoing angiogenesis, we subjected mice to hypoxia-induced proliferative retinopathy. As a result, PDGF-B(+/-) mice developed twice as many new blood vessels as their wild-type littermates. We conclude that retinal capillary coverage with pericytes is crucial for the survival of endothelial cells, particularly under stress conditions such as diabetes. At high vascular endothelial growth factor levels, such as those in the retinopathy of prematurity model, pericyte deficiency leads to reduced inhibition of endothelial proliferation in vivo.

Angiopoietin-2 causes pericyte dropout in the normal retina: evidence for involvement in diabetic retinopathy.

Pericyte loss is an early pathologic feature of diabetic retinopathy, consistently present in retinae of diabetic humans and animals. Because pericyte recruitment and endothelial cell survival are controlled, in part, by the angiopoietin/Tie2 ligand/receptor system, we studied the expression of angiopoietin-2 and -1 in relation to the evolution of pericyte loss in diabetic rat retinae, using quantitative retinal morphometry, and in retinae from mice with heterozygous angiopoietin deficiency (Ang-2 LacZ knock-in mice). Finally, recombinant angiopoietin-2 was injected into eyes of nondiabetic rats, and pericyte numbers were quantitated in retinal capillaries. Angiopoietin-1 protein was present in the normal maturing retina and was upregulated 2.5-fold in diabetic retinae over 3 months of diabetes. In contrast, angiopoietin-2 protein was consistently upregulated more than 30-fold in the retinae of diabetic rats, preceding the onset of pericyte loss. Heterozygous angiopoietin-2 deficiency completely prevented diabetes-induced pericyte loss and reduced the number of acellular capillary segments. Injection of angiopoietin-2 into the eyes of normal rats induced a dose-dependent pericyte loss. These data show that upregulation of angiopoietin-2 plays a critical role in the loss of pericytes in the diabetic retina.

Time- and cell type-specific induction of platelet-derived growth factor receptor-beta during cerebral ischemia.

During cerebral ischemia, angiogenesis occurs inside and around the infarcted area. The growth of new blood vessels may contribute to a better outcome after stroke due to accelerated and increased delivery of nutrients and oxygen to the ischemic tissue. The platelet-derived growth factor (PDGF)-B/PDGF receptor (PDGFR)-beta system, hitherto thought to contribute mainly to neuroprotection, may also support angiogenesis and vascular remodeling by mediating interactions of endothelial cells with pericytes after cerebral ischemia. While platelet-derived growth factor (PDGF)-B and its receptor PDGFR-beta are essential factors for the recruitment of pericytes to brain capillaries during embryonic development, their role in blood vessel maturation during cerebral ischemia is not clear. The aim of the present study was to investigate the time course and location of PDGF-B and PDGFR-beta expression in a mouse model of focal cerebral ischemia. In contrast to the early and continuous induction of PDGF-B, PDGFR-beta mRNA was specifically upregulated in vascular structures in the infarcted area 48 h after occlusion of the middle cerebral artery. Immunohistology and confocal microscopy analysis revealed the specific upregulation of PDGFR-beta on blood vessels and suggested expression mainly on pericytes. Our results imply PDGFR-beta as a key factor in vascular remodeling during stroke and suggest that the pleiotropic functions of PDGF-B may be regulated via the expression of its receptor. Influencing the PDGF system therapeutically might improve angiogenesis, cellular protection, and edema inhibition.

Genetic Modeling of PIM Proteins in Cancer: Proviral Tagging and Cooperation with Oncogenes, Tumor Suppressor Genes, and Carcinogens.

The PIM proteins, which were initially discovered as proviral insertion sites in Moloney-murine leukemia virus infection, are a family of highly homologous serine/threonine kinases that have been reported to be overexpressed in hematological malignancies and solid tumors. The PIM proteins have also been associated with metastasis and overall treatment responses and implicated in the regulation of apoptosis, metabolism, the cell cycle, and homing and migration, which makes these proteins interesting targets for anti-cancer drug discovery. The use of retroviral insertional mutagenesis and refined approaches such as complementation tagging has allowed the identification of myc, pim, and a third group of genes (including bmi1 and gfi1) as complementing genes in lymphomagenesis. Moreover, mouse modeling of human cancer has provided an understanding of the molecular pathways that are involved in tumor initiation and progression at the physiological level. In particular, genetically modified mice have allowed researchers to further elucidate the role of each of the Pim isoforms in various tumor types. PIM kinases have been identified as weak oncogenes because experimental overexpression in lymphoid tissue, prostate, and liver induces tumors at a relatively low incidence and with a long latency. However, very strong synergistic tumorigenicity between Pim1/2 and c-Myc and other oncogenes has been observed in lymphoid tissues. Mouse models have also been used to study whether the inhibition of specific PIM isoforms is required to prevent carcinogen-induced sarcomas, indicating that the absence of Pim2 and Pim3 greatly reduces sarcoma growth and bone invasion; the extent of this effect is similar to that observed in the absence of all three isoforms. This review will summarize some of the animal models that have been used to understand the isoform-specific contribution of PIM kinases to tumorigenesis.

Conditional transgenic expression of PIM1 kinase in prostate induces inflammation-dependent neoplasia.

The Pim proteins are a family of highly homologous protein serine/threonine kinases that have been found to be overexpressed in cancer. Elevated levels of Pim1 kinase were first discovered in human leukemia and lymphomas. However, more recently Pim1 was found to be increased in solid tumors, including pancreatic and prostate cancers, and has been proposed as a prognostic marker. Although the Pim kinases have been identified as oncogenes in transgenic models, they have weak transforming abilities on their own. However, they have been shown to greatly enhance the ability of other genes or chemical carcinogens to induce tumors. To explore the role of Pim1 in prostate cancer, we generated conditional Pim1 transgenic mice, expressed Pim1 in prostate epithelium, and analyzed the contribution of PIM1 to neoplastic initiation and progression. Accordingly, we explored the effect of PIM1 overexpression in 3 different settings: upon hormone treatment, during aging, and in combination with the absence of one Pten allele. We have found that Pim1 overexpression increased the severity of mouse prostate intraepithelial neoplasias (mPIN) moderately in all three settings. Furthermore, Pim1 overexpression, in combination with the hormone treatment, increased inflammation surrounding target tissues leading to pyelonephritis in transgenic animals. Analysis of senescence induced in these prostatic lesions showed that the lesions induced in the presence of inflammation exhibited different behavior than those induced in the absence of inflammation. While high grade prostate preneoplastic lesions, mPIN grades III and IV, in the presence of inflammation did not show any senescence markers and demonstrated high levels of Ki67 staining, untreated animals without inflammation showed senescence markers and had low levels of Ki67 staining in similar high grade lesions. Our data suggest that Pim1 might contribute to progression rather than initiation in prostate neoplasia.

Exploring the gain of function contribution of AKT to mammary tumorigenesis in mouse models.

Elevated expression of AKT has been noted in a significant percentage of primary human breast cancers, mainly as a consequence of the PTEN/PI3K pathway deregulation. To investigate the mechanistic basis of the AKT gain of function-dependent mechanisms of breast tumorigenesis, we explored the phenotype induced by activated AKT transgenes in a quantitative manner. We generated several transgenic mice lines expressing different levels of constitutively active AKT in the mammary gland. We thoroughly analyzed the preneoplastic and neoplastic mammary lesions of these mice and correlated the process of tumorigenesis to AKT levels. Finally, we analyzed the impact that a possible senescent checkpoint might have in the tumor promotion inhibition observed, crossing these lines to mammary specific p53(R172H) mutant expression, and to p27 knock-out mice. We analyzed the benign, premalignant and malignant lesions extensively by pathology and at molecular level analysing the expression of proteins involved in the PI3K/AKT pathway and in cellular senescence. Our findings revealed an increased preneoplastic phenotype depending upon AKT signaling which was not altered by p27 or p53 loss. However, p53 inactivation by R172H point mutation combined with myrAKT transgenic expression significantly increased the percentage and size of mammary carcinoma observed, but was not sufficient to promote full penetrance of the tumorigenic phenotype. Molecular analysis suggest that tumors from double myrAKT;p53(R172H) mice result from acceleration of initiated p53(R172H) tumors and not from bypass of AKT-induced oncogenic senescence. Our work suggests that tumors are not the consequence of the bypass of senescence in MIN. We also show that AKT-induced oncogenic senescence is dependent of pRb but not of p53. Finally, our work also suggests that the cooperation observed between mutant p53 and activated AKT is due to AKT-induced acceleration of mutant p53-induced tumors. Finally, our work shows that levels of activated AKT are not essential in the induction of benign or premalignant tumors, or in the cooperation of AKT with other tumorigenic signal such as mutant p53, once AKT pathway is activated, the relative level of activity seems not to determine the phenotype.

Mouse models to decipher the PI3K signaling network in human cancer.

Deregulation of the PI3K pathway is common in human cancer. The basic players in this pathway are the kinases PI3K and AKT and the phosphatase PTEN. This review will summarize some of the key animal models that have helped us understand this signaling network and its contribution to tumorigenesis. Recently, great advances in cancer mouse models have been achieved [1]. While germline deletion often affects the development of the organism and can result in embryonic lethality, conditional knockout mouse models offer the possibility of inducing gene deletion in the adult organism. Another useful strategy involves the inactivation of enzyme function by introducing small mutations in the gene sequence, thereby maintaining the protein in an inactivated state and mimicking protein inhibition with drugs. Combining tissue-specific expression of PI3K and AKT with a secondary oncogenic event, for example, leads to the development of specific tumors. Such models are more accurate for growth studies of human tumors than those involving xenograft tumors, due to the interconnection of the tumors with blood vessels. These studies will improve the pharmacological analysis of drug candidates. With the discovery of oncogenic mutations in members of the PI3K pathway, mouse lines harboring these mutations are being developed in order to imitate the molecular features of a human tumor. Using these models for drug testing may enable more accurate prediction of the effects that a specific drug will have on a patient.

Activation of phosphatidylinositol 3-kinase by membrane localization of p110alpha predisposes mammary glands to neoplastic transformation.

Phosphatidylinositol 3-kinases (PI3K) constitute important regulators of various signaling pathways with relevance in cancer. Enhanced activation of p110alpha, the catalytic subunit of PI3K, was found in a high proportion of many human tumor types. We generated a mouse model in which PI3K is activated by forced recruitment of p110alpha to the membrane. Different transgenic lines expressing myristoylated p110alpha protein under the control of the epithelial-specific mouse mammary tumor virus promoter were selected according to different levels of PI3K activity and characterized. Delayed mammary gland involution and morphologic changes of the mammary ducts could be detected in young transgenic female mice. These changes were more pronounced in old animals, especially in mutiparous females, in which we observed increased ductal branching, alveolar hyperplasia, and intraductal neoplasia. We also observed a small percentage of mammary tumors. Crosses of myrp110alpha transgenic mice with heterozygous p53(+/-) knockout mice resulted in neither enhanced tumorigenesis nor in a stronger mammary gland phenotype. However, the CDK4 activating mutation (R24C) lead to increased tumorigenesis in transgenic myrp110alpha mice, emphasizing the postulated perturbation of the interaction of the CDK4/Rb/E2F cascade and the PI3K signaling in many human cancers. Interestingly, in tumors of myrp110alpha transgenic mice, we observed an increased phosphorylation of the estrogen receptor-alpha, a typical feature of human breast cancer. The model presented here will help to discover additional factors which influence the progression of preneoplastic lesions to tumors in the mammary gland and to explore antitumor therapies based on PI3K or estrogen receptor-alpha pathway inhibition.

The PTEN/PI3K/AKT signalling pathway in cancer, therapeutic implications.

PTEN/PI3K/AKT constitutes an important pathway regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth. PTEN is a dual protein/lipid phosphatase which main substrate is the phosphatidyl-inositol,3,4,5 triphosphate (PIP3), the product of PI3K. Increase in PIP3 recruits AKT to the membrane where it is activated by other kinases also dependent on PIP3. Many components of this pathway have been described as causal forces in cancer. PTEN activity is lost by mutations, deletions or promoter methylation silencing at high frequency in many primary and metastatic human cancers. Germ line mutations of PTEN are found in several familial cancer predisposition syndromes. Activating mutations which have been reported for PI3K and AKT, in tumours are able to confer tumourigenic properties in several cellular systems. Additionally, the binding of PI3K to oncogenic ras is essential for the transforming properties of ras. In summary, the data strongly support the view of the PTEN/PI3K/AKT pathway as an important target for drug discovery.

PTEN, more than the AKT pathway.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/phosphatidylinositol 3-kinase (PI3K)/AKT constitute an important pathway regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth. PTEN is a dual protein/lipid phosphatase and its main substrate phosphatidyl-inositol 3,4,5 triphosphate (PIP3) is the product of PI3K. Increase in PIP3 recruits AKT to the membrane where is activated by other kinases also dependent on PIP3. Many components of this pathway have been described as causal forces in cancer. PTEN activity is lost by mutations, deletions or promoter methylation silencing at high frequency in many primary and metastatic human cancers. Germ line mutations of PTEN are found in several familial cancer predisposition syndromes. Recently, many activating mutations in the PI3KCA gene (coding for the p110alpha catalytic subunit of PI3K) have been described in human tumors. Activation of PI3K and AKT are reported to occur in breast, ovarian, pancreatic, esophageal and other cancers. Genetically modified mice confirm these PTEN activities. Tissue-specific deletions of PTEN usually provoke cancer. Moreover, an absence of PTEN cooperates with an absence of p53 to promote cancer. However, we have observed very different results with the expression of activated versions of AKT in several tissues. Activated AKT transgenic lines do not develop tumors in breast or prostate tissues and do not cooperate with an absence of p53. This data suggest that an AKT-independent mechanism contributes to PTEN tumorigenesis. Crosses with transgenic mice expressing possible PTEN targets indicate that neither cyclin D1 nor p53 are these AKT-independent targets. However, AKT is more than a passive bridge toward PTEN tumorigenesis, since its expression not only allows but also enforces and accelerates the tumorigenic process in combination with other oncogenes.

Mst1, RanBP2 and eIF4G are new markers for in vivo PI3K activation in murine and human prostate.

Phosphatidylinositol 3-kinases (PI3Ks) constitute important regulators of signaling pathways. The PIK3CA gene encoding the p110-alpha catalytic subunit represents one of the highly mutated oncogenes identified in human cancer. Here, we report new markers for in vivo PI3K activation in prostate. To that end, we used a transgenic mouse line, which expresses a constitutively active p110-alpha subunit in the epithelial cells of the prostate. The activity of the PI3K pathway in the prostate was proven by assessing the phosphorylation of the PI3K direct target AKT1 and of the mTOR target eukaryotic translation initiation factor 4G (eIF4G). To establish also transcriptional ('late') targets of the PI3K pathway, we tested two genes, Mst1 and RanBP2, which we recently described as transcriptional targets of the growth factor platelet-derived growth factor-beta. We show that the levels of both proteins are elevated in transgenic animals. Additionally, we describe that the phosphorylation of AKT and eIF4G, as well as the elevation of the Mst1 and RanBP2 protein levels, can be inhibited in vivo in transgenic animals by the PI3K inhibitor LY294002. Finally, we performed human tissue microarray experiments with the four markers. Since they define overlapping but not identical subsets of the tested tissue panel, a combination of all four markers might lead to a more accurate diagnosis of the status of the PI3K-signaling cascade in cancer patients.

Mice expressing myrAKT1 in the mammary gland develop carcinogen-induced ER-positive mammary tumors that mimic human breast cancer.

AKT1/PKB is a serine/threonine protein kinase that regulates biological processes such as proliferation, apoptosis and growth in a variety of cell types. To assess the oncogenic capability of an activated form of AKT in vivo we have generated several transgenic mouse lines that overexpress in the mammary epithelium the murine Akt1 gene modified with a myristoylation signal, which renders active this protein by localizing it to the plasma membrane. We demonstrate that expression of myristoylated AKT in the mammary glands increases the susceptibility of these mice to the induction of mammary tumors of epithelial origin by the carcinogen 9,10-dimethyl-1,2 benzanthracene (DMBA). We have found that while carcinogen-treated wild-type mice show mostly mammary tumors of sarcomatous origin, AKT transgenic mice treated with DMBA developed mainly adenocarcinoma or adenosquamous tumors, all of them displaying activated AKT. We analyzed other possible molecular alterations cooperating with AKT and found that neither Ras nor beta-catenin/Wnt pathways seemed altered nor p53 mutated. We have found that 100% of mammary DMBA-induced tumors and benign lesions in myrAKT mice are estrogen receptor (ERalpha)-positive and are more frequent than in wild-type littermates. These data show that AKT activation cooperates with deregulation of the estrogen receptor in the DMBA-induced mammary tumorigenesis model and recapitulate two characteristics of some human breast tumors. Thus, our model might provide a preclinical relevant model system to study the role of AKT and ERalpha in breast tumorigenesis and the response of mammary gland tumors to chemotherapeutics.

Identification of transcripts specific for physiological gene activation by platelet-derived growth factor (PDGF)-B in intact brain tissue.

Platelet-derived growth factor-B (PDGF-B) and its receptors play essential roles in the complex process of blood vessel maturation and they therefore constitute promising targets for therapeutic strategies against blood vessel-related diseases. Additionally, they are involved in the autocrine stimulation of tumor cells and have been suggested to regulate tumor stroma fibroblasts. Our study aimed to identify genes that are regulated directly by PDGF-B, or indirectly via the recruitment of perivascular cells, in the context of an intact tissue. We used a subtractive cloning technique to compare gene transcription in the brains of wild-type (WT) mice and syngenic mice deficient of PDGF-B leading to a defect in the recruitment of perivascular cells. The resulting 147 differentially expressed sequences contained early and late PDGF-B target genes, and genes implicated in blood vessel maturation-related pathways. Additionally, gene clusters for specific biological processes such as cell migration and intracellular transport were identified. Of eight randomly selected sequences, six were found expressed in cultured cells of mesenchymal origin, two of them inducible by exogenous PDGF-BB. The collection of cDNA presented here provides insights into the changes provoked by the removal of one growth factor of a complete tissue and might be the basis for the identification of novel players in the complex process of blood vessel maturation.

Transcription profiling of platelet-derived growth factor-B-deficient mouse embryos identifies RGS5 as a novel marker for pericytes and vascular smooth muscle cells.

All blood capillaries consist of endothelial tubes surrounded by mural cells referred to as pericytes. The origin, recruitment, and function of the pericytes is poorly understood, but the importance of these cells is underscored by the severe cardiovascular defects in mice genetically devoid of factors regulating pericyte recruitment to embryonic vessels, and by the association between pericyte loss and microangiopathy in diabetes mellitus. A general problem in the study of pericytes is the shortage of markers for these cells. To identify new markers for pericytes, we have taken advantage of the platelet-derived growth factor (PDGF)-B knockout mouse model, in which developing blood vessels in the central nervous system are almost completely devoid of pericytes. Using cDNA microarrays, we analyzed the gene expression in PDGF-B null embryos in comparison with corresponding wild-type embryos and searched for down-regulated genes. The most down-regulated gene present on our microarray was RGS5, a member of the RGS family of GTPase-activating proteins for G proteins. In situ hybridization identified RGS5 expression in brain pericytes, and in pericytes and vascular smooth muscle cells in certain other, but not all, locations. Absence of RGS5 expression in PDGF-B and PDGFR beta-null embryos correlated with pericyte loss in these mice. Residual RGS5 expression in rare pericytes suggested that RGS5 is a pericyte marker expressed independently of PDGF-B/R beta signaling. With RGS5 as a proof-of-principle, our data demonstrate the usefulness of microarray analysis of mouse models for abnormal pericyte development in the identification of new pericyte-specific markers.