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1.  Pericyte-specific expression of PDGF beta receptor in mouse models with normal and deficient PDGF beta receptor signaling 
Background
Pericytes are integral members of the neurovascular unit. Using mouse models lacking endothelial-secreted platelet derived growth factor-B (PDGF-B) or platelet derived growth factor receptor beta (PDGFRβ) on pericytes, it has been demonstrated that PDGF-B/PDGFRβ interactions mediate pericyte recruitment to the vessel wall in the embryonic brain regulating the development of the cerebral microcirculation and the blood-brain barrier (BBB). Relatively little is known, however, about the roles of PDGF-B/PDGFRβ interactions and pericytes in the adult brain in part due to a lack of adequate and/or properly characterized experimental models. To address whether genetic disruption of PDGFRβ signaling would result in a pericyte-specific insult in adult mice, we studied the pattern and cellular distribution of PDGFRβ expression in the brain in adult control mice and F7 mice that express two hypomorphic Pdgfrβ alleles containing seven point mutations in the cytoplasmic domain of PDGFRβ that impair downstream PDGFRβ receptor signaling.
Results
Using dual fluorescent in situ hybridization, immunofluorescent staining for different cell types in the neurovascular unit, and a fluorescent in situ proximity ligation assay to visualize molecular PDGF-B/PDGFRβ interactions on brain tissue sections, we show for the first time that PDGFRβ is exclusively expressed in pericytes, and not in neurons, astrocytes or endothelial cells, in the adult brain of control 129S1/SvlmJ mice. PDGFRβ co-localized only with well-established pericyte markers such as Chondroitin Sulfate Proteoglycan NG2 and the xLacZ4 transgenic reporter. We next confirm pericyte-specific PDGFRβ expression in the brains of F7 mutants and show that these mice are viable in spite of substantial 40-60% reductions in regional pericyte coverage of brain capillaries.
Conclusions
Our data show that PDGFRβ is exclusively expressed in pericytes in the adult 129S1/Sv1mJ and F7 mouse brain. Moreover, our findings suggest that genetic disruption of PDGFRβ signaling results in a pericyte-specific insult in adult F7 mutants and will not exert a primary effect on neurons because PDGFRβ is not expressed in neurons of the adult 129S1/SvlmJ and F7 mouse brain. Therefore, mouse models with normal and deficient PDGFRβ signaling on a 129S1/SvlmJ background may effectively be used to deduce the specific roles of pericytes in maintaining the cerebral microcirculation and BBB integrity in the adult and aging brain as well as during neurodegenerative and brain vascular disorders.
doi:10.1186/1750-1326-5-32
PMCID: PMC2936891  PMID: 20738866
2.  Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis 
The Journal of Clinical Investigation  2013;123(8):3436-3445.
Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/TIE2 interaction during sepsis is a potential therapeutic target.
doi:10.1172/JCI66549
PMCID: PMC3726157  PMID: 23863629
3.  Host-Derived Angiopoietin-2 Affects Early Stages of Tumor Development and Vessel Maturation but Is Dispensable for Later Stages of Tumor Growth 
Cancer research  2009;69(4):1324-1333.
The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence. Angiopoietin-2 (Ang-2) is prominently up-regulated in the host-derived vasculature of most tumors, making it an attractive candidate for antiangiogenic intervention. Yet, the net outcome of Ang-2 functions on tumor angiogenesis is believed to be contextual depending on the local cytokine milieu. Correspondingly, Ang-2 manipulatory therapies have been shown to exert protumorigenic as well as antitumorigenic effects. To clarify the role of Ang-2 for angiogenesis and tumor growth in a definite genetic experimental setting, the present study was aimed at comparatively studying the growth of different tumors in wild-type and Ang-2–deficient mice. Lewis lung carcinomas, MT-ret melanomas, and B16F10 melanomas all grew slower in Ang-2–deficient mice. Yet, tumor growth in wild-type and Ang-2–deficient mice dissociated during early stages of tumor development, whereas tumor growth rates during later stages of primary tumor progression were similar. Analysis of the intratumoral vascular architecture revealed no major differences in microvessel density and perfusion characteristics. However, diameters of intratumoral microvessels were smaller in tumors grown in Ang-2–deficient mice, and the vasculature had an altered pattern of pericyte recruitment and maturation. Ang-2–deficient tumor vessels had higher pericyte coverage indices. Recruited pericytes were desmin and NG2 positive and predominately α-smooth muscle actin negative, indicative of a more mature pericyte phenotype. Collectively, the experiments define the role of Ang-2 during tumor angiogenesis and establish a better rationale for combination therapies involving Ang-2 manipulatory therapies.
doi:10.1158/0008-5472.CAN-08-3030
PMCID: PMC3514474  PMID: 19208839
4.  Id Proteins Regulate Capillary Repair and Perivascular Cell Proliferation following Ischemia-Reperfusion Injury 
PLoS ONE  2014;9(2):e88417.
Acute kidney injury (AKI) results in microvascular damage that if not normally repaired, may lead to fibrosis. The Id1 and 3 proteins have a critical role in promoting angiogenesis during development, tumor growth and wound repair by functioning as dominant negative regulators of bHLH transcription factors. The goal of this study was to determine if Id proteins regulate microvascular repair and remodeling and if increased Id1 expression results in decreased capillary loss following AKI. The effect of changes in Id expression in vivo was examined using Id1−/−, Id3RFP/+ (Id1/Id3 KO) and Tek (Tie2)-rtTA, TRE-lacz/TRE Id1 (TRE Id1) mice with doxycycline inducible endothelial Id1 and β-galactosidase expression. Id1 and 3 were co-localized in endothelial cells in normal adult kidneys and protein levels were increased at day 3 following ischemia-reperfusion injury (IRI) and contralateral nephrectomy. Id1/Id3 KO mice had decreased baseline capillary density and pericyte coverage and increased tubular damage following IRI but decreased interstitial cell proliferation and fibrosis compared with WT littermates. No compensatory increase in kidney size occurred in KO mice resulting in increased creatinine compared with WT and TRE Id1 mice. TRE Id1 mice had no capillary rarefaction within 1 week following IRI in comparison with WT littermates. TRE Id1 mice had increased proliferation of PDGFRβ positive interstitial cells and medullary collagen deposition and developed capillary rarefaction and albuminuria at later time points. These differences were associated with increased Angiopoietin 1 (Ang1) and decreased Ang2 expression in TRE Id1 mice. Examination of gene expression in microvascular cells isolated from WT, Id1/Id3 KO and TRE Id1 mice showed increased Ang1 and αSMA in Id1 overexpressing cells and decreased pericyte markers in cells from KO mice. These results suggest that increased Id levels following AKI result in microvascular remodeling associated with increased fibrosis.
doi:10.1371/journal.pone.0088417
PMCID: PMC3917915  PMID: 24516656
5.  Early Vessel Destabilization Mediated by Angiopoietin-2 and Subsequent Vessel Maturation via Angiopoietin-1 Induce Functional Neovasculature after Ischemia 
PLoS ONE  2013;8(4):e61831.
Background
We assessed whether Angiopoietin-2 (Ang2), a Tie2 ligand and partial antagonist of Angiopoietin-1 (Ang1), is required for early vessel destabilization during postischemic angiogenesis, when combined with vascular growth factors.
Methods
In vitro, matrigel co-cultures assessed endothelial-cell tube formation and pericyte recruitment after stimulation of VEGF-A, Apelin (APLN), Ang1 with or without Ang2. In a murine hindlimb ischemia model, adeno-associated virus (rAAV, 3×1012 virusparticles) transduction of VEGF-A, APLN and Ang1 with or without Ang2 (continuous or early expression d0-3) was performed intramuscularly (d-14). Femoral artery ligation was performed at d0, followed by laser doppler perfusion meassurements (LDI) 7 and 14. At d7 (early timepoint) and d14 (late timepoint), histological analysis of capillary/muscle fiber ratio (CMF-R, PECAM-1) and pericyte/capillary ratio (PC-R, NG2) was performed.
Results
In vitro, VEGF-A, APLN and Ang1 induced ring formation, but only APLN and Ang1 recruited pericytes. Ang2 did not affect tube formation by APLN, but reduced pericyte recruitment after APLN or Ang1 overexpression. In vivo, rAAV.VEGF-A did not alter LDI-perfusion at d14, consistent with an impaired PC-R despite a rise in CMF-R. rAAV.APLN improved perfusion at d14, with or without continuous Ang2, increasing CMF-R and PC-R. rAAV.Ang1 improved perfusion at d14, when combined with rAAV.Ang2 (d0-3), accompanied by an increased CMF-R and PC-R.
Conclusion
The combination of early vessel destabilization (Ang2 d0-3) and continuous Ang1 overexpression improves hindlimb perfusion, pointing to the importance of early vessel destabilization and subsequent vessel maturation for enhanced therapeutic neovascularization.
doi:10.1371/journal.pone.0061831
PMCID: PMC3628915  PMID: 23613948
6.  Blood–spinal cord barrier pericyte reductions contribute to increased capillary permeability 
The blood–spinal cord barrier (BSCB) regulates molecular exchange between blood and spinal cord. Pericytes are presumed to be important cellular constituents of the BSCB. However, the regional abundance and vascular functions of spinal cord pericytes have yet to be determined. Utilizing wild-type mice, we show that spinal cord pericyte capillary coverage and number compared with the brain regions are reduced most prominently in the anterior horn. Regional pericyte variations are highly correlated with: (1) increased capillary permeability to 350 Da, 40,000 Da, and 150,000 Da, but not 2,000,000 Da fluorescent vascular tracers in cervical, thoracic, and lumbar regions and (2) diminished endothelial zonula occludens-1 (ZO-1) and occludin tight junction protein expression. Pericyte-deficient mutations (PdgfrβF7/F7 mice) resulted in additional pericyte reductions in spinal cord capillaries leading to overt BSCB disruption to serum proteins, accumulation in motor neurons of cyotoxic thrombin and fibrin and motor neuron loss. Barrier disruption in perciyte-deficient mice coincided with further reductions in ZO-1 and occludin. These data suggest that pericytes contribute to proper function of the BSCB at the capillary level. Regional reductions in spinal cord pericytes may provide a cellular basis for heightened spinal cord barrier capillary permeability and motor neuron loss.
doi:10.1038/jcbfm.2012.113
PMCID: PMC3463878  PMID: 22850407
blood–brain barrier; capillaries; endothelium; pericytes; vascular biology
7.  Pericytes Derived from Adipose-Derived Stem Cells Protect against Retinal Vasculopathy 
PLoS ONE  2013;8(5):e65691.
Background
Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy.
Methodology/Principal Findings
We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection).
Conclusions/Significance
ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-β1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.
doi:10.1371/journal.pone.0065691
PMCID: PMC3669216  PMID: 23741506
8.  Pericytopathy: Oxidative Stress and Impaired Cellular Longevity in the Pancreas and Skeletal Muscle in Metabolic Syndrome and Type 2 Diabetes 
The pericyte's role has been extensively studied in retinal tissues of diabetic retinopathy; however, little is known regarding its role in such tissues as the pancreas and skeletal muscle. This supportive microvascular mural cell plays an important and novel role in cellular and extracellular matrix remodeling in the pancreas and skeletal muscle of young rodent models representing the metabolic syndrome and type 2 diabetes mellitus (T2DM). Transmission electron microscopy can be used to evaluate these tissues from young rodent models of insulin resistance and T2DM, including the transgenic Ren2 rat, db/db obese insulin resistantߞT2DM mouse, and human islet amyloid polypeptide (HIP) rat model of T2DM. With this method, the earliest pancreatic remodeling change was widening of the islet exocrine interface and pericyte hypercellularity, followed by pericyte differentiation into islet and pancreatic stellate cells with early fibrosis involving the islet exocrine interface and interlobular interstitium. In skeletal muscle there was a unique endothelial capillary connectivity via elongated longitudinal pericyte processes in addition to pericyte to pericyte and pericyte to myocyte cellcell connections allowing for paracrine communication. Initial pericyte activation due to moderate oxidative stress signaling may be followed by hyperplasia, migration and differentiation into adult mesenchymal cells. Continued robust oxidative stress may induce pericyte apoptosis and impaired cellular longevity. Circulating antipericyte autoantibodies have recently been characterized, and may provide a screening method to detect those patients who are developing pericyte loss and are at greater risk for the development of complications of T2DM due to pericytopathy and rarefaction. Once detected, these patients may be offered more aggressive treatment strategies such as early pharmacotherapy in addition to lifestyle changes targeted to maintaining pericyte integrity. In conclusion, we have provided a review of current knowledge regarding the pericyte and novel ultrastructural findings regarding its role in metabolic syndrome and T2DM.
doi:10.4161/oxim.3.5.13653
PMCID: PMC3154033  PMID: 21150342
9.  Modulation of Vascular Cell Function by Bim Expression 
Apoptosis of vascular cells, including pericytes and endothelial cells, contributes to disease pathogenesis in which vascular rarefaction plays a central role. Bim is a proapoptotic protein that modulates not only apoptosis but also cellular functions such as migration and extracellular matrix (ECM) protein expression. Endothelial cells and pericytes each make a unique contribution to vascular formation and function although the details require further delineation. Here we set out to determine the cell autonomous impact of Bim expression on retinal endothelial cell and pericyte function using cells prepared from Bim deficient (Bim−/−) mice. Bim−/− endothelial cells displayed an increased production of ECM proteins, proliferation, migration, adhesion, and VEGF expression but, a decreased eNOS expression and nitric oxide production. In contrast, pericyte proliferation decreased in the absence of Bim while migration, adhesion, and VEGF expression were increased. In addition, we demonstrated that the coculturing of either wild-type or Bim−/− endothelial cells with Bim−/− pericytes diminished their capillary morphogenesis. Thus, our data further emphasizes the importance of vascular cell autonomous regulatory mechanisms in modulation of vascular function.
doi:10.1155/2013/297537
PMCID: PMC3833316  PMID: 24288535
10.  Antibody-Mediated Retinal Pericyte Injury: Implications for Diabetic Retinopathy 
Purpose.
To test the hypothesis that autoantibodies against retinal pericytes could develop in diabetic retinopathy, and that these autoantibodies could induce retinal pericyte dysfunction/death via complement.
Methods.
Human primary retinal pericytes cultured in media containing normal (5 mM) or high (30 mM) glucose concentrations were incubated with normal human sera in the presence of a retinal pericyte-reactive antibody, then their viability was assessed by a BCECF-based cytotoxicity assay, and their function was assessed by a T-cell proliferation assay. The pericytes were also analyzed by RT-PCR and flow cytometry to detect CD38, an established diabetes-associated cell surface autoantigen. The potential of the anti-CD38 antibodies in inducing pericyte cellular injury was evaluated using the same cytotoxicity assays. In addition, autoantibody-mediated cytotoxicity in mouse retinal pericytes sensitized by sera from mice with developing diabetic retinopathy or control normal mice were also studied.
Results.
Retinal pericyte–reactive antibodies induced cellular damage by activating complement in the serum. The antibody-injured pericytes had reduced efficacy in inhibiting T cells. Hyperglycemic culture conditions rendered pericytes more susceptible to antibody-mediated attack. CD38 was expressed in retinal pericytes, and upregulated by TNF-α and IFN-γ, and anti-CD38 antibodies induced pericyte cytotoxicity. Retinal pericytes sensitized with sera from chronic diabetic mice suffered significantly augmented cytotoxicity compared with those sensitized with sera from the control mice.
Conclusions.
The autoantibody-initiated complement activation could be a mechanism underlying the loss of function, and eventually, death of retinal pericytes in diabetic patients, suggesting that inhibiting complement activation could be a novel therapeutic approach.
Data presented in this report suggest that autoantibodies against retinal pericyte cell surface antigens induce pericyte cytotoxicity through complement, which could contribute to the development of diabetic retinopathy.
doi:10.1167/iovs.12-10010
PMCID: PMC3419006  PMID: 22786897
11.  Frontiers in Research: Chronic Kidney Diseases: The pivotal role of pericytes in kidney fibrosis 
SUMMARY
Kidney pericytes were recently identified as collagen-Iα1 producing cells in healthy kidney, but the developmental, physiological and pathological roles of kidney pericytes remain poorly understood. Pericytes are stromal-derived cells that envelop, and have intimate connections with adjacent capillary endothelial cells (ECs). Recent studies in eye and brain have revealed that pericytes are crucial for angiogenesis, vascular stability and vessel integrity.In response to kidney injury, pericytes promptly migrate away from the capillary wall into the interstitial space. Here, pericytes are activated and differentiate into scar-forming myofibroblasts. In the absence of pericytes, peritubular capillaries are destabilized leading to vascular regression. Consequently, capillary loss and fibrosis following kidney injury are intimately linked and hinge centrally around pericyte detachment from ECs.Kinetic mathematical modeling demonstrated that pericytes are the major source of myofibroblasts in fibrotic kidney. Comprehensive genetic fate mapping studies of nephron epithelia or kidney stroma has demonstrated that epithelial cells do not migrate outside of epithelial compartment to become myofibroblasts rather that interstitial pericytes are progenitors of scar-forming myofibroblasts. Bidirectional signaling between pericytes and ECs is necessary for pericyte detachment from peritubular capillaries.In the present review, we summarize the pathologically vital roles of kidney pericytes in fibrosis including our new findings. The study of kidney pericytes and endothelial-pericyte crosstalk will identify novel therapeutic targets for currently incurable chronic kidney diseases.
doi:10.1111/j.1440-1681.2011.05531.x
PMCID: PMC3124592  PMID: 21517936
capillary rarefaction; chronic kidney diseases; endothelial cells; epithelial-to-mesenchymal transition (EMT); fibrosis; kidney pericytes; kidney injury; myofibroblasts; peritubular capillary
12.  Blood–spinal cord barrier breakdown and pericyte reductions in amyotrophic lateral sclerosis 
Acta Neuropathologica  2012;125(1):111-120.
The blood–brain barrier and blood–spinal cord barrier (BSCB) limit the entry of plasma components and erythrocytes into the central nervous system (CNS). Pericytes play a key role in maintaining blood–CNS barriers. The BSCB is damaged in patients with amyotrophic lateral sclerosis (ALS). Moreover, transgenic ALS rodents and pericyte-deficient mice develop BSCB disruption with erythrocyte extravasation preceding motor neuron dysfunction. Here, we studied whether BSCB disruption with erythrocyte extravasation and pericyte loss are present in human ALS. We show that 11 of 11 cervical cords from ALS patients, but 0 of 5 non-neurodegenerative disorders controls, possess perivascular deposits of erythrocyte-derived hemoglobin and hemosiderin typically 10–50 μm in diameter suggestive of erythrocyte extravasation. Immunostaining for CD235a, a specific marker for erythrocytes, confirmed sporadic erythrocyte extravasation in ALS, but not controls. Quantitative analysis revealed a 3.1-fold increase in perivascular hemoglobin deposits in ALS compared to controls showing hemoglobin confined within the vascular lumen, which correlated with 2.5-fold increase in hemosiderin deposits (r = 0.82, p < 0.01). Spinal cord parenchymal accumulation of plasma-derived immunoglobulin G, fibrin and thrombin was demonstrated in ALS, but not controls. Immunostaining for platelet-derived growth factor receptor-β, a specific marker for CNS pericytes, indicated a 54 % (p < 0.01) reduction in pericyte number in ALS patients compared to controls. Pericyte reduction correlated negatively with the magnitude of BSCB damage as determined by hemoglobin abundance (r = −0.75, p < 0.01). Thus, the BSCB disruption with erythrocyte extravasation and pericyte reductions is present in ALS. Whether similar findings occur in motor cortex and affected brainstem motor nuclei remain to be seen.
doi:10.1007/s00401-012-1039-8
PMCID: PMC3535352  PMID: 22941226
Amyotrophic lateral sclerosis; Pericytes; Vascular injury; Blood–brain barrier; Blood–spinal cord barrier
13.  Effect of cartilage oligomeric matrix protein angiopoietin-1 on peripheral nerves in db/db diabetic mice 
Background: Vascular and inflammatory processes have been reported to be factors in the pathogenesis of diabetic neuropathy. Angiopoietin-1 (Ang1) plays essential roles in regulating vascular growth, development, maturation, permeability, and inflammation.
Objective: The aim of this study was to investigate the effect of cartilage oligomeric matrix protein (COMP)-Ang1, which is a soluble, stable, potent Ang1 variant, on peripheral nerves in db/db diabetic mice.
Methods: The db/db diabetic mice were randomized into 2 groups based on their weight and glucose level and treated with recombinant adenovirus (Ade), expressing either COMP-Ang1 or the β-galactosidase gene (LacZ) (control), for 8 weeks. Immunohistochemistry was performed using a polyclonal antibody of antiprotein gene product and a secondary antibody. Intraepidermal nerve fiber density (IENFD) was quantified as nerve fiber abundance per unit length of epidermis (IENF/mm). In addition, the total capillary length (TCL) per unit length of epidermis was summed (mm/mm2). All slides were coded and the capillary length and the number of nerve fibers were calculated by a blinded observer.
Results: Ten diabetic db/db mice (mean [SD] weight, 38.7 [1.95] g) were randomized to receive Ade-COMP-Ang1 or Ade-LacZ. IENFD was significantly greater in the Ade-COMP-Ang1 group compared with the Ade-LacZ group (mean [SD] 8.95 [3.30] vs 3.57 [0.73]/mm; P < 0.05). TCL was also significantly greater in the Ade-COMP-Ang1 group (2.79 [0.99] vs 2.04 [0.58] mm/mm2; P < 0.05). Compared with baseline, fasting blood glucose concentration after 8 weeks of treatment decreased significantly more in the Ade-COMP-Ang1 group than in the Ade-LacZ group (489 [45] to 361 [81] vs 495 [48] to 521 [70] mg/dL; P < 0.05).
Conclusions: These results suggest that Ade-COMP-Ang1 might have had proliferative effects on peripheral nerve and cutaneous capillaries in this small animal study. Further investigation of the metabolic effect, target site, and related mediator of COMP-Ang1 is needed.
doi:10.1016/j.curtheres.2008.08.002
PMCID: PMC3969925  PMID: 24692811
COMP-angiopoietin-1; db/db diabetic mice; peripheral neuropathy
14.  DEFICIENCY IN MURAL VASCULAR CELLS COINCIDES WITH BLOOD-BRAIN BARRIER DISRUPTION IN ALZHEIMER’S DISEASE 
Neurovascular dysfunction contributes to Alzheimer’s disease (AD). Cerebrovascular abnormalities and blood-brain barrier (BBB) damage have been shown in AD. The BBB dysfunction can lead to leakage of potentially neurotoxic plasma components in brain that may contribute to neuronal injury. Pericytes are integral in maintaining the BBB integrity. Pericyte-deficient mice develop a chronic BBB damage preceding neuronal injury. Moreover, loss of pericytes was associated with BBB breakdown in patients with amyotrophic lateral sclerosis. Here, we demonstrate a decrease in mural vascular cells in AD, and show that pericyte number and coverage in the cortex and hippocampus of AD subjects compared to neurologically-intact controls are reduced by 59% and 60% (p<0.01), and 32 and 33% (p<0.01), respectively. An increase in extravascular immunoglobulin G and fibrin deposition correlated with reductions in pericyte coverage in AD cases compared to controls; the Pearson’s correlation coefficient r for the magnitude of BBB breakdown to IgG and fibrin versus reduction in pericyte coverage was − 0.96 (p<0.01) and − 0.81 (p<0.01) in the cortex, respectively, and − 0.86 (p<0.01) and − 0.98 (p<0.01) in the hippocampus, respectively. Thus, deficiency in mural vascular cells may contribute to disrupted vascular barrier properties and resultant neuronal dysfunction during AD pathogenesis.
doi:10.1111/bpa.12004
PMCID: PMC3628957  PMID: 23126372
Pericytes; Alzheimer’s disease; blood-brain barrier
15.  Pericytes: brain-immune interface modulators 
The premise that the central nervous system is immune-privileged arose from the fact that direct contact between immune and nervous cells is hindered by the blood–brain barrier. However, the blood–brain barrier also comprises the interface between the immune and nervous systems by secreting chemo-attractant molecules and by modulating immune cell entry into the brain. The majority of published studies on the blood–brain barrier focus on endothelial cells (ECs), which are a critical component, but not the only one; other cellular components include astroglia, microglia, and pericytes. Pericytes are poorly studied in comparison with astrocytes or ECs; they are mesenchymal cells that can modify their ultrastructure and gene expression in response to changes in the central nervous system microenvironment. Pericytes have a unique synergistic relationship with brain ECs in the regulation of capillary permeability through secretion of cytokines, chemokines, nitric oxide, matrix metalloproteinases, and by means of capillary contraction. Those pericyte manifestations are related to changes in blood–brain barrier permeability by an increase in endocytosis-mediated transport and by tight junction disruption. In addition, recent reports demonstrate that pericytes control the migration of leukocytes in response to inflammatory mediators by up-regulating the expression of adhesion molecules and releasing chemo-attractants; however, under physiological conditions they appear to be immune-suppressors. Better understanding of the immune properties of pericytes and their participation in the effects of brain infections, neurodegenerative diseases, and sleep loss will be achieved by analyzing pericyte ultrastructure, capillary coverage, and protein expression. That knowledge may provide a mechanism by which pericytes participate in the maintenance of the proper function of the brain-immune interface.
doi:10.3389/fnint.2013.00080
PMCID: PMC3887314  PMID: 24454281
pericytes; blood–brain barrier; immune response; inflammation; cytokines; REM sleep loss; brain endothelial cell; tight junction disruption
16.  Notch Signaling Functions in Retinal Pericyte Survival 
Purpose.
Pericytes, the vascular cells that constitute the outer layer of capillaries, have been shown to have a crucial role in vascular development and stability. Loss of pericytes precedes endothelial cell dysfunction and vascular degeneration in small-vessel diseases, including diabetic retinopathy. Despite their clinical relevance, the cellular pathways controlling survival of retinal pericytes remain largely uncharacterized. Therefore, we investigated the role of Notch signaling, a master regulator of cell fate decisions, in retinal pericyte survival.
Methods.
A coculture system of ligand-dependent Notch signaling was developed using primary cultured retinal pericytes and a mesenchymal cell line derived from an inducible mouse model expressing the Delta-like 1 Notch ligand. This model was used to examine the effect of Notch activity on pericyte survival using quantitative PCR (qPCR) and a light-induced cell death assay. The effect of Notch gain- and loss-of-function was analyzed in monocultures of retinal pericytes using antibody arrays to interrogate the expression of apoptosis-related proteins.
Results.
Primary cultured retinal pericytes differentially expressed key molecules of the Notch pathway and displayed strong expression of canonical Notch/RBPJK (recombination signal-binding protein 1 for J-kappa) downstream targets. A gene expression screen using gain- and loss-of-function approaches identified genes relevant to cell survival as downstream targets of Notch activity in retinal pericytes. Ligand-mediated Notch activity protected retinal pericytes from light-induced cell death.
Conclusions.
Our results have identified signature genes downstream of Notch activity in retinal pericytes and suggest that tight regulation of Notch signaling is crucial for pericyte survival.
Mono- and coculture systems were used to uncover a role for Notch signaling, a master regulator of cell fate decisions, in pericyte survival.
doi:10.1167/iovs.14-14046
PMCID: PMC4139112  PMID: 25015359
diabetic retinopathy; survival; notch signaling; coculture; pericyte; small-vessel
17.  Pericyte loss influences Alzheimer-like neurodegeneration in mice 
Nature Communications  2013;4:2932.
Pericytes are cells in the blood–brain barrier that degenerate in Alzheimer’s disease (AD), a neurological disorder associated with neurovascular dysfunction, abnormal elevation of amyloid β-peptide (Aβ), tau pathology and neuronal loss. Whether pericyte degeneration can influence AD-like neurodegeneration and contribute to disease pathogenesis remains, however, unknown. Here we show that in mice overexpressing Aβ-precursor protein, pericyte loss elevates brain Aβ40 and Aβ42 levels and accelerates amyloid angiopathy and cerebral β-amyloidosis by diminishing clearance of soluble Aβ40 and Aβ42 from brain interstitial fluid prior to Aβ deposition. We further show that pericyte deficiency leads to the development of tau pathology and an early neuronal loss that is normally absent in Aβ-precursor protein transgenic mice, resulting in cognitive decline. Our data suggest that pericytes control multiple steps of AD-like neurodegeneration pathogenic cascade in Aβ-precursor protein-overexpressing mice. Therefore, pericytes may represent a novel therapeutic target to modify disease progression in AD.
Pericytes are cells in the blood–brain barrier that degenerate with the onset of Alzheimer's disease. Here, Sagare et al. show that pericyte loss contributes to disease onset by promoting amyloid-beta accumulation, tau pathology and early loss of neuronal cells.
doi:10.1038/ncomms3932
PMCID: PMC3945879  PMID: 24336108
18.  Pericytes synthesize renin 
World Journal of Nephrology  2013;2(1):11-16.
AIM: To investigate renin expression in pericytes during normal kidney development and after deletion of angiotensinogen, the precursor for all angiotensins.
METHODS: We examined the distribution of renin expressing cells by immunoshistochemistry in the interstitial compartment of wild type (WT) and angiotensinogen deficient (AGT -/-) mice at different developmental stages from embryonic day 18 (E18: WT, n = 4; AGT -/-, n = 5) and at day 1 (P1: WT, n = 5; AGT -/-, n = 5), 5 (P5: WT, n = 7; AGT -/-, n = 8), 10 (P10: WT, n = 3; AGT -/-, n = 5), 21 (P21: WT, n = 7; AGT -/-, n = 5), 45 (P45: WT, n = 3; AGT -/-, n = 3), and 70 (P70: WT, n = 2; AGT -/-, n = 2) of postnatal life. We quantified the number of pericytes positive for renin at all the developmental stages mentioned above and compared the results of AGT -/- mice to their WT counterparts.
RESULTS: In WT mice, renal interstitial pericytes synthesize renin in early life supporting a lineage relationship with renin cells in the vasculature. The number of pericytes positive for renin per area of 0.32 mm2 (density) in WT mice was maintained from fetal life till weaning age (E18 = 4.25 ± 0.63, P1 = 3.75 ± 0.48, P5 = 3.75 ± 0.48, P10 = 4 ± 0.71, P21 = 3.8 ± 0.58) and markedly decreased in adult life (P45 = 1.2 ± 0.37, P70 = 0.8 ± 0.20). On the other hand, in AGT -/- mice the density of pericytes expressing renin was not significantly different from WT mice at E18 and P1: E18 = 5.75 ± 0.50 vs 4.25 ± 0.63 (P = 0.106), P1 = 9.25 ± 3.50 vs 3.75 ± 0.48 (P = 0.175) but significantly increased from P5 till P70: P5 = 38.25 ± 5 vs 3.75 ± 0.48 (P = 0.0004), P10 = 173 ± 7.50 vs 4 ± 0.70 (P = 5.24567 × 10-7), P21 = 83 ± 6.70 vs 3.8 ± 0.58 (P = 2.97358 × 10-6), P45 = 49 ± 3.50 vs 1.2 ± 0.37 (P = 8.18274 x 10-7) and P70 = 17.8 ± 2.30 vs 0.8 ± 0.20 (P = 3.51151 × 10-5). The AGT -/- mice showed a marked increase in the number of pericytes per field studied starting from P5, reaching its peak at P10, and then a gradually decreasing until P70.
CONCLUSION: Interstitial pericytes synthesize renin during development and the number of renin-expressing pericytes increases in response to a homeostatic threat imposed early in life such as lack of angiotensinogen.
doi:10.5527/wjn.v2.i1.11
PMCID: PMC3782206  PMID: 24175260
Interstitium; Homeostasis; Angiotensinogen; Kidney; Renin angiotensin system; Development; Angiotensin deficiency; Gene deletion
19.  Human Pericytes for Ischemic Heart Repair 
Stem cells (Dayton, Ohio)  2013;31(2):305-316.
Human microvascular pericytes (CD146+/34−/45−/56−) contain multipotent precursors and repair/regenerate defective tissues, notably skeletal muscle. However, their ability to repair the ischemic heart remains unknown. We investigated the therapeutic potential of human pericytes, purified from skeletal muscle, for treating ischemic heart disease and mediating associated repair mechanisms in mice. Echocardiography revealed that pericyte transplantation attenuated left ventricular dilatation and significantly improved cardiac contractility, superior to CD56+ myogenic progenitor transplantation, in acutely infarcted mouse hearts. Pericyte treatment substantially reduced myocardial fibrosis and significantly diminished infiltration of host inflammatory cells at the infarct site. Hypoxic pericyte-conditioned medium suppressed murine fibroblast proliferation and inhibited macrophage proliferation in vitro. High expression by pericytes of immunoregulatory molecules, including IL-6, LIF, COX-2 and HMOX-1, was sustained under hypoxia, except for MCP-1. Host angiogenesis was significantly increased. Pericytes supported microvascular structures in vivo and formed capillary-like networks with/without endothelial cells in three-dimensional co-cultures. Under hypoxia, pericytes dramatically increased expression of VEGF-A, PDGF-β, TGF-β1 and corresponding receptors while expression of bFGF, HGF, EGF, and Ang-1 was repressed. The capacity of pericytes to differentiate into and/or fuse with cardiac cells was revealed by GFP-labeling, though to a minor extent. In conclusion, intramyocardial transplantation of purified human pericytes promotes functional and structural recovery, attributable to multiple mechanisms involving paracrine effects and cellular interactions.
doi:10.1002/stem.1285
PMCID: PMC3572307  PMID: 23165704
pericytes; angiogenesis; immunomodulation; myocardial infarction; stem cell therapy
20.  Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging 
Neuron  2010;68(3):409-427.
SUMMARY
Pericytes play a key role in the development of cerebral microcirculation. The exact role of pericytes in the neurovascular unit in the adult brain and during brain aging remains, however, elusive. Using adult viable pericyte-deficient mice, we show that pericyte loss leads to brain vascular damage by two parallel pathways: (1) reduction in brain microcirculation causing diminished brain capillary perfusion, cerebral blood flow and cerebral blood flow responses to brain activation which ultimately mediates chronic perfusion stress and hypoxia, and (2) blood-brain barrier breakdown associated with brain accumulation of serum proteins and several vasculotoxic and/or neurotoxic macromolecules ultimately leading to secondary neuronal degenerative changes. We show that age-dependent vascular damage in pericyte-deficient mice precedes neuronal degenerative changes, learning and memory impairment and the neuroinflammatory response. Thus, pericytes control key neurovascular functions that are necessary for proper neuronal structure and function, and pericytes loss results in a progressive age-dependent vascular-mediated neurodegeneration.
doi:10.1016/j.neuron.2010.09.043
PMCID: PMC3056408  PMID: 21040844
21.  SPARC promotes pericyte recruitment via inhibition of endoglin-dependent TGF-β1 activity 
The Journal of Cell Biology  2011;193(7):1305-1319.
SPARC prevents endoglin association with αV integrin, which blocks the activation of TGF-β signaling and promotes pericyte migration to nascent blood vessels.
Pericytes migrate to nascent vessels and promote vessel stability. Recently, we reported that secreted protein acidic and rich in cysteine (SPARC)–deficient mice exhibited decreased pericyte-associated vessels in an orthotopic model of pancreatic cancer, suggesting that SPARC influences pericyte behavior. In this paper, we report that SPARC promotes pericyte migration by regulating the function of endoglin, a TGF-β1 accessory receptor. Primary SPARC-deficient pericytes exhibited increased basal TGF-β1 activity and decreased cell migration, an effect blocked by inhibiting TGF-β1. Furthermore, TGF-β–mediated inhibition of pericyte migration was dependent on endoglin and αV integrin. SPARC interacted directly with endoglin and reduced endoglin interaction with αV integrin. SPARC deficiency resulted in endoglin-mediated blockade of pericyte migration, aberrant association of endoglin in focal complexes, an increase in αV integrins present in endoglin immunoprecipitates, and enhanced αV integrin–mediated activation of TGF-β. These results demonstrate that SPARC promotes pericyte migration by diminishing TGF-β activity and identify a novel function for endoglin in controlling pericyte behavior.
doi:10.1083/jcb.201011143
PMCID: PMC3216331  PMID: 21708981
22.  Enhancing Microvascular Formation And Vessel Maturation Through Temporal Control Over Multiple Pro-Angiogenic And Pro-Maturation Factors 
Biomaterials  2013;34(36):10.1016/j.biomaterials.2013.08.007.
Therapeutic stimulation of angiogenesis to re-establish blood flow in ischemic tissues offers great promise as a treatment for patients suffering from cardiovascular disease or trauma. Since angiogenesis is a complex, multi-step process, different signals may need to be delivered at appropriate times in order to promote a robust and mature vasculature. The effects of temporally regulated presentation of pro-angiogenic and pro-maturation factors were investigated in vitro and in vivo in this study. Pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) cooperatively promoted endothelial sprouting and pericyte detachment in a three-dimensional in vitro EC-pericyte co-culture model. Pro-maturation factors platelet-derived growth factor B (PDGF) and angiopoietin 1 (Ang1) inhibited the early stages of VEGF- and Ang2-mediated angiogenesis if present simultaneously with VEGF and Ang2, but promoted these behaviors if added subsequently to the pro-angiogenesis factors. VEGF and Ang2 were also found to additively enhance microvessel density in a subcutaneous model of blood vessel formation, while simultaneously administered PDGF/Ang1 inhibited microvessel formation. However, a temporally controlled scaffold that released PDGF and Ang1 at a delay relative to VEGF/Ang2 promoted both vessel maturation and vascular remodeling without inhibiting sprouting angiogenesis. Our results demonstrate the importance of temporal control over signaling in promoting vascular growth, vessel maturation and vascular remodeling. Delivering multiple growth factors in combination and sequence could aid in creating tissue engineered constructs and therapies aimed at promoting healing after acute wounds and in chronic conditions such as diabetic ulcers and peripheral artery disease.
doi:10.1016/j.biomaterials.2013.08.007
PMCID: PMC3811005  PMID: 23972477
23.  Activation of PKCδ and SHP1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy 
Nature Medicine  2009;15(11):1298-1306.
Cellular apoptosis induced by hyperglycemia occurs in many vascular cells and is critical to initiate diabetic pathologies. In the retina, pericyte apoptosis, the most specific vascular pathology attributed to hyperglycemia, is linked to the loss of PDGF actions due to unknown mechanisms. Our study demonstrated that hyperglycemia persistently activated PKCδ and p38α MAPK to increase the expression of a novel target, SHP-1, leading to PDGF receptor-β dephosphorylation and actions, and increased pericyte apoptosis, independent of NF-κB. These findings were also observed in diabetic mouse retinas, which were not reversed by achieving normoglycemia with insulin. Unlike diabetic controls, diabetic Prkcd−/− mice did not exhibit p38α MAPK/SHP-1 activation, PDGF resistance or acellular capillaries. Since PKCδ/p38α MAPK/SHP-1 activation are also induced in the brain pericytes and renal cortex by diabetes, these findings have elucidated a new pathway by which hyperglycemia can induce PDGF resistance and increase vascular cell apoptosis to cause diabetic vascular complications.
doi:10.1038/nm.2052
PMCID: PMC3290906  PMID: 19881493
24.  Pericytes from Infantile Hemangioma Display Pro-angiogenic Properties and Dysregulated Angiopoietin-1 
OBJECTIVE
Infantile hemangioma (IH) is a rapidly growing vascular tumor affecting newborns. It is composed of immature endothelial cells and pericytes that proliferate into a disorganized mass of blood vessels. We isolated pericytes from IH (Hem-pericytes) to test our hypothesis that Hem-pericytes are unable to stabilize blood vessels.
METHODS AND RESULTS
We injected pericytes in vivo, in combination with endothelial cells, and found that Hem-pericytes formed more microvessels compared to control retinal pericytes. We thereby analyzed pro-angiogenic properties of the Hem-pericytes. They grew fast in vitro, and were unable to stabilize endothelial cell growth and migration, and expressed high levels of VEGF-A compared to retinal pericytes. Hem-pericytes from proliferating phase IH showed lower contractility in vitro, compared to Hem-pericytes from the involuting phase and retinal pericytes. Consistent with a diminished ability to stabilize endothelium, Angiopoietin 1 (ANGPT1) was reduced in Hem-pericytes compared to retinal pericytes. Normal retinal pericytes in which ANGPT1 was silenced produced conditioned medium that stimulated endothelial cell proliferation and migration.
CONCLUSIONS
We report the first successful isolation of patient-derived pericytes from IH tissue. Hem-pericytes exhibited pro-angiogenic properties and low levels of ANGPT1, consistent with a diminished ability to stabilize blood vessels in IH.
doi:10.1161/ATVBAHA.112.300929
PMCID: PMC3573237  PMID: 23288163
infantile hemangioma; pericytes; angiogenesis; VEGF-A; endothelial colony forming cells; Angiopoietin1
25.  Effects of Angiopoietin-2-Blocking Antibody on Endothelial Cell–Cell Junctions and Lung Metastasis 
Background
Angiopoietin-2 (Ang2), a ligand for endothelial TEK (Tie2) tyrosine kinase receptor, is induced in hypoxic endothelial cells of tumors, where it promotes tumor angiogenesis and growth. However, the effects of Ang2 on tumor lymphangiogenesis and metastasis are poorly characterized.
Methods
We addressed the effect of Ang2 on tumor progression and metastasis using systemic Ang2 overexpression in mice carrying tumor xenografts, endothelium-specific overexpression of Ang2 in VEC-tTA/Tet-OS-Ang2 transgenic mice implanted with isogenic tumors, and administration of Ang2-blocking antibodies to tumor-bearing immunodeficient mice. Fisher's exact test was used for analysis of metastasis occurrence, and repeated measures one-way analysis of variance was used for the analysis of primary tumor growth curves. Unpaired t test was used for all other analyses. All statistical tests were two-sided.
Results
Adenoviral expression of Ang2 increased lymph node and lung metastasis in tumor xenografts. The metastatic burden in the lungs was increased in transgenic mice in which Ang2 expression was induced specifically in the vascular endothelium (tumor burden per grid, VEC-tTA/Tet-OS-Ang2 mice [n = 5] vs control mice [n = 4]: 45.23 vs 12.26 mm2, difference = 32.67 mm2, 95% confidence interval = 31.87 to 34.07, P < .001). Ang2-blocking antibodies reduced lymph node and lung metastasis, as well as tumor lymphangiogenesis, and decreased tumor cell homing to the lungs after intravenous injection. In the lung metastases, Ang2 overexpression decreased endothelial integrity, whereas the Ang2-blocking antibodies improved endothelial cell–cell junctions and basement membrane contacts of metastasis-associated lung capillaries. At the cellular level, the Ang2-blocking antibodies induced the internalization of Ang2-Tie2 receptor complexes from endothelial cell–cell junctions in endothelial–tumor cell cocultures.
Conclusion
Our results indicate that blocking Ang2 inhibits metastatic dissemination in part by enhancing the integrity of endothelial cell–cell junctions.
doi:10.1093/jnci/djs009
PMCID: PMC3309130  PMID: 22343031

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