Pericyte-endothelial Cell Interactions Research Articles

The longevity-associated BPIFB4 gene supports cardiac function and vascularization in ageing cardiomyopathy.

The ageing heart naturally incurs a progressive decline in function and perfusion that available treatments cannot halt. However, some exceptional individuals maintain good health until the very late stage of their life due to favourable gene-environment interaction. We have previously shown that carriers of a longevity-associated variant (LAV) of the BPIFB4 gene enjoy prolonged health spans and lesser cardiovascular complications. Moreover, supplementation of LAV-BPIFB4 via an adeno-associated viral vector improves cardiovascular performance in limb ischaemia, atherosclerosis, and diabetes models. Here, we asked whether the LAV-BPIFB4 gene could address the unmet therapeutic need to delay the heart's spontaneous ageing. Immunohistological studies showed a remarkable reduction in vessel coverage by pericytes in failing hearts explanted from elderly patients. This defect was attenuated in patients carrying the homozygous LAV-BPIFB4 genotype. Moreover, pericytes isolated from older hearts showed low levels of BPIFB4, depressed pro-angiogenic activity, and loss of ribosome biogenesis. LAV-BPIFB4 supplementation restored pericyte function and pericyte-endothelial cell interactions through a mechanism involving the nucleolar protein nucleolin. Conversely, BPIFB4 silencing in normal pericytes mimed the heart failure pericytes. Finally, gene therapy with LAV-BPIFB4 prevented cardiac deterioration in middle-aged mice and rescued cardiac function and myocardial perfusion in older mice by improving microvasculature density and pericyte coverage. We report the success of the LAV-BPIFB4 gene/protein in improving homeostatic processes in the heart's ageing. These findings open to using LAV-BPIFB4 to reverse the decline of heart performance in older people.

The role of pericytes in cardiac ageing and disease

Abstract Introduction Cardiac disease induces remodelling which can include fibrosis, cardiomyocyte hypertrophy, cardiac dilation, and finally can lead to heart failure. Age is one of the main risk factors of cardiovascular disease and induces heart remodelling, in particular, it has profound effects on the microcirculation. Pericytes are microvascular mural cells involved in the maintenance of stability and homeostasis of the vascular network. Although the phenotypes that arise from cardiac remodelling have been well studied in cardiomyocytes, endothelial cells and fibroblasts, the effect of aging on pericytes remain largely unknown. Purpose The purpose of this study is to characterise pericyte responses to cardiac ageing and disease in order to determine the therapeutic potential of these mural cells to reverse, or at least reduce, structural remodelling. Methods We have studied 12-week-old and 18-month-old mice. We have performed histological analysis and single-nucleus-RNA-sequencing (snRNAseq). For our in vitro experiments we have used primary human pericytes. Results Age affects the structure of the microcirculation in the heart. Pericyte coverage is reduced and capillary diameter is increased. Gene ontology analysis of differentially expressed genes in pericytes revealed an upregulation of genes related to filopodia and actin cytoskeleton, while a reduction of genes related to focal adhesion in the pericytes of the aged heart. Interestingly, we detected a downregulation of Regulator of G-protein signalling 5 (RGS5), a repressor of GPCRs signalling. RGS5 knockdown induces a contractile, pro-inflammatory and pro-fibrotic gene expression profile reducing pericytes proliferation and migration. Mechanistically, we show that RGS5 post-transcriptionally regulates PDGFRβ, a crucial tyrosine kinase receptor for pericyte-endothelial cell interaction. RGS5 knockdown reduces the expression of the receptor at the protein level, but not at the gene expression level and furthermore reduces the phosphorylation of AKT, a downstream signal of PDGFRβ activity. Furthermore, we have identified that T-Box Transcription Factor 20 (Tbx20), a cardiogenic transcription factor, is enriched in aged pericytes. Silencing and upregulation studies have revealed that Tbx20 is a repressor of PDGFRB, the gene that encodes for PDGFRβ, and that it controls pericyte adhesion. Conclusions Together, these observations have identified RGS5 and Tbx20 as crucial key players maintaining pericyte function in the aged heart. We propose that RGS5 and Tbx20 regulate pericyte function by controlling PDGFRβ signalling and cellular proliferation, adhesion and migration. Given the importance of pericytes in keeping vessel homeostasis, maintaining or recovering pericyte function in context of cardiac stress would be a potential approach to reduce the malignant effects of cardiac remodelling in the aged heart. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SFB1366 (DFG)

Investigating Atypical Inflammatory Signaling in Vascular Pericytes as Potential Target for Controlling Blood‐Retinal‐Barrier (BRB) Inflammation in Diabetic Retinopathy

Pericytes are master regulators of vascular homeostasis and play key roles in the progression of many vasculopathies, including neurovascular inflammation and diabetic retinopathy. During inflammation, pericytes can be activated by many signals, leading to pericyte induced proinflammatory signaling and pericyte migration away from the vasculature leaving endothelial tubules exposed and fragile. Despite a clear role for G protein‐coupled receptors (GPCR) in the regulation of vascular endothelial inflammation, GPCR control of pericytes has been understudied. Furthermore, pericytes represent a promising candidate for blocking inflammatory responses that could mitigate the onset and progression of neurovascular disease. Our prior studies revealed an atypical inflammatory pathway in endothelial cells, regulated by GPCR induced autoactivation of the mitogen‐activated protein kinase (MAPK) p38 pathway. This Atypical inflammatory pathway has not been studied in pericytes and is a promising therapeutic target. Our central hypothesis is that neurovascular inflammation in the brain and eye is controlled by atypical p38 signaling in vascular pericytes. To investigate this we are initially using primary human brain pericytes. We show that that thrombin, histamine and prostaglandin E2 (PGE2) can activate p38 via the atypical p38 signaling pathway in primary human pericytes and that a peptide inhibitor of atypical p38 signaling, blocks PGE2 dependent p38 activation, suppressing proinflammatory cytokine expression. Furthermore we are currently using single cell morphometric analysis to characterize heterogeneous pericyte responses to proinflammatory stimuli. This will enable the correlation of morphological variations to proinflammatory roles in T‐cell modulation, cytokine regulation, and migration. These studies are revealing novel insights into GPCR dependent control of pericytes and will be extrapolated into studies investigating pericyte‐endothelial cell interactions and the dysregulation of the blood‐retinal‐barrier during diabetic retinopathy. Our current studies focus on defining this role and in understanding how the neurovascular unit in the eye is regulated through atypical signaling, and whether inhibition of atypical signaling can protect the retina from damage. Thus, these studies represent a novel approach to regulate vascular p38 activity and pro‐inflammatory signaling in retinal disease.

Hexokinase 2-driven glycolysis in pericytes activates their contractility leading to tumor blood vessel abnormalities

Defective pericyte-endothelial cell interaction in tumors leads to a chaotic, poorly organized and dysfunctional vasculature. However, the underlying mechanism behind this is poorly studied. Herein, we develop a method that combines magnetic beads and flow cytometry cell sorting to isolate pericytes from tumors and normal adjacent tissues from patients with non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC). Pericytes from tumors show defective blood vessel supporting functions when comparing to those obtained from normal tissues. Mechanistically, combined proteomics and metabolic flux analysis reveals elevated hexokinase 2(HK2)-driven glycolysis in tumor pericytes, which up-regulates their ROCK2-MLC2 mediated contractility leading to impaired blood vessel supporting function. Clinically, high percentage of HK2 positive pericytes in blood vessels correlates with poor patient overall survival in NSCLC and HCC. Administration of a HK2 inhibitor induces pericyte-MLC2 driven tumor vasculature remodeling leading to enhanced drug delivery and efficacy against tumor growth. Overall, these data suggest that glycolysis in tumor pericytes regulates their blood vessel supporting role.

Pericytes on placental capillaries in terminal villi preferentially cover endothelial junctions in regions furthest away from the trophoblast

IntroductionPericytes are a common feature in the placental microvasculature but their roles are not well understood. Pericytes may provide physical or endocrine support for endothelium and in some tissues mediate vasoconstriction. MethodsThis study uses serial block-face scanning electron microscopy (SBFSEM) to generate three-dimensional (3D) reconstructions of placental pericytes of the terminal villi and transmission electron microscopy (TEM) to study pericyte endothelial cell interactions. The proportion of endothelial cell junctions covered by pericytes was determined. ResultsThe detailed 3D models of placental pericytes show pericyte structure at a new level of detail. Placental pericytes have many fingers extending from the cell body which can span multiple capillary branches. The proportion of endothelial cell-cell junctions covered by pericytes was significantly higher than pericyte coverage of capillary endothelium as a whole (endothelium: 14%, junctions: 43%, p < 0.0001). However, the proportion of endothelial cell-cell junctions covered by pericytes in regions adjacent to trophoblast was reduced compared to regions >3 μm away from trophoblast (27% vs 62% respectively, p < 0.001). No junctional complexes were observed connecting pericytes and endothelial cells but there were regions of cell membrane with features suggestive of intercellular adhesions. DiscussionThese data suggest that the localisation of pericytes on the villous capillary is not random but organised in relation to both endothelial junctions and the location of adjacent trophoblast. This further suggests that pericyte coverage may favour capillary permeability in regions that are most important for exchange, but limit capillary permeability in other regions.

Dimethyl-2-oxoglutarate improves redox balance and mitochondrial function in muscle pericytes of individuals with diabetes mellitus

Aims/hypothesisTreatment of vascular complications of diabetes remains inadequate. We reported that muscle pericytes (MPs) from limb muscles of vascular patients with diabetes mellitus display elevated levels of oxidative stress causing a dysfunctional phenotype. Here, we investigated whether treatment with dimethyl-2-oxoglutarate (DM-2OG), a tricarboxylic acid cycle metabolite with antioxidant properties, can restore a healthy metabolic and functional phenotype.MethodsMPs were isolated from limb muscles of diabetes patients with vascular disease (D-MPs) and from non-diabetic control participants (ND-MPs). Metabolic status was assessed in untreated and DM-2OG-treated (1 mmol/l) cells using an extracellular flux analyser and anion-exchange chromatography–mass spectrometry (IC-MS/MS). Redox status was measured using commercial kits and IC-MS/MS, with antioxidant and metabolic enzyme expression assessed by quantitative RT-PCR and western blotting. Myogenic differentiation and proliferation and pericyte–endothelial interaction were assessed as functional readouts.ResultsD-MPs showed mitochondrial dysfunction, suppressed glycolytic activity and reduced reactive oxygen species-buffering capacity, but no suppression of antioxidant systems when compared with ND-MP controls. DM-2OG supplementation improved redox balance and mitochondrial function, without affecting glycolysis or antioxidant systems. Nonetheless, this was not enough for treated D-MPs to regain the level of proliferation and myogenic differentiation of ND-MPs. Interestingly, DM-2OG exerted a positive effect on pericyte–endothelial cell interaction in the co-culture angiogenesis assay, independent of the diabetic status.Conclusions/interpretationThese novel findings support the concept of using DM-2OG supplementation to improve pericyte redox balance and mitochondrial function, while concurrently allowing for enhanced pericyte–endothelial crosstalk. Such effects may help to prevent or slow down vasculopathy in skeletal muscles of people with diabetes.Graphical abstract

Sleep loss disrupts pericyte-brain endothelial cell interactions impairing blood-brain barrier function

Sleep loss in the rat increases blood-brain barrier permeability to circulating molecules by disrupting interendothelial tight junctions. Despite the description of the ultrastructure of cerebral microvessels and the evidence of an apparent pericyte detachment from capillary wall in sleep restricted rats the effect of sleep loss on pericytes is unknown. Here we characterized the interactions between pericytes and brain endothelial cells after sleep loss using male Wistar rats. Animals were sleep-restricted 20 h daily with 4 h sleep recovery for 10 days. At the end of the sleep restriction, brain microvessels (MVs) were isolated from cerebral cortex and hippocampus and processed for Western blot and immunocytochemistry to evaluate markers of pericyte-endothelial cell interaction (connexin 43, PDGFR-β), tight junction proteins, and proinflammatory mediator proteins (MMP9, A2A adenosine receptor, CD73, NFκB). Sleep restriction reduced PDGFR-β and connexin 43 expression in MVs; in addition, scanning electron microscopy micrographs showed that pericytes were detached from capillary walls, but did not undergo apoptosis (as depicted by a reduced active caspase-3 expression). Sleep restriction also decreased tight junction protein expression in MVs and increased BBB permeability to low- and high-molecular weight tracers in in vivo permeability assays. Those alterations seemed to depend on a low-grade inflammatory status as reflected by the increased expression of phosphorylated NFκB and A2A adenosine receptor in brain endothelial cells from the sleep-restricted rats. Our data show that pericyte-brain endothelial cell interaction is altered by sleep restriction; this evidence is essential to understand the role of sleep in regulating blood-brain barrier function.

Abstract 708: Platelet Derived Growth Factor-Beta Communication Axis is Critical for Mediating Pericyte-Endothelial Cell Interactions and Angiogenic Support in Combination Therapy of Human Umbilical Cord Perivascular Cells and Endothelial Progenitor Cells

Introduction: First Trimester Human Umbilical Cord Perivascular Cells (FTM HUCPVC) have higher cardiovascular regenerative potential compared to older sources of MSC. We designed a cell combination therapy with FTM HUCPVC and endothelial progenitor cells (EPC) to enhance angiogenesis after MI. However, the mechanism of PVC-EPC interaction is unclear. Hypothesis: Platelet derived growth factor beta receptor (PDGFB-R) expression of HUCPVC mediates homing and integration with endothelial networks and required for supporting neovasculature. Methods: Expression of pericyte markers PDGFB-R and CD146 of xeno-free expanded (HPL 2.5%) FTM and term HUCPVC was analyzed by flow cytometry. PDGFB-driven trans-ECM migration was assessed by Corning Biocoat Invasion assay with 1-100 ng/ml PDGFB. siRNA for PDGFB-R, CD146 and scrambled control was used with lipofectamine for 72 hours. Silencing was confirmed by qPCR and flow cytometry. 10% HPL was used for PDGFB-R desensitization. HUCPVC of all treatment groups were co-cultured with EPC and rat aortic rings on Matrigel TM . Cell migration, endothelial adhesion and network properties were quantified. Statistics: one-way ANOVA N=5 Results: PDGFB-R and CD146 expression and endothelial network augmenting function of FTM HUCPVC was significantly higher than of term HUCPVC. PDGFB dependent migration of FTM HUCPVC was disabled by both ligand (&lt;10%) and siRNA-based (&lt;5%) downregulation of PDGFB-R. Fluorescence microscopy (24 h) showed limited homing and endothelial interactions of PDGFB-R-reduced FTM HUCPVC. PDGFB-R-reduced FTM HUCPVC had significantly decreased network growth and nodes ( p &lt;0.0001) compared to control groups (day3). In EPC co-cultures, PDGFB-R-reduced FTM HUCPVC had limited endothelial interactions and coverage compared to control FTM HUCPVC. PDGFB-R-reduced FTM HUCPVC treated networks had significantly thinner ( p &lt;0.001) and shorter tubules ( p &lt;0.001) compared to control FTM HUCPVC groups. Down-regulation of C146 did not alter FTM HUCPVC pericyte properties. Conclusions: PDGFB-R is crucial for the homing and endothelial support of HUCPVC. This interaction is critical in vascular cell combination therapies and PDGFB-R expression can be a novel criterion for cardiovascular cell therapy.

Pericyte-Derived Dickkopf2 Regenerates Damaged Penile Neurovasculature Through an Angiopoietin-1-Tie2 Pathway.

Penile erection requires well-coordinated interactions between vascular and nervous systems. Penile neurovascular dysfunction is a major cause of erectile dysfunction (ED) in patients with diabetes, which causes poor response to oral phosphodiesterase-5 inhibitors. Dickkopf2 (DKK2), a Wnt antagonist, is known to promote angiogenesis. Here, using DKK2-Tg mice or DKK2 protein administration, we demonstrate that the overexpression of DKK2 in diabetic mice enhances penile angiogenesis and neural regeneration and restores erectile function. Transcriptome analysis revealed that angiopoietin-1 and angiopoietin-2 are target genes for DKK2. Using an endothelial cell-pericyte coculture system and ex vivo neurite sprouting assay, we found that DKK2-mediated juxtacrine signaling in pericyte-endothelial cell interactions promotes angiogenesis and neural regeneration through an angiopoietin-1-Tie2 pathway, rescuing erectile function in diabetic mice. The dual angiogenic and neurotrophic effects of DKK2, especially as a therapeutic protein, will open new avenues to treating diabetic ED.

Dual roles of endothelial FGF-2\u2013FGFR1\u2013PDGF-BB and perivascular FGF-2\u2013FGFR2\u2013PDGFR\u03b2 signaling pathways in tumor vascular remodeling

Perivascular cells are important cellular components in the tumor microenvironment (TME) and they modulate vascular integrity, remodeling, stability, and functions. Here we show using mice models that FGF-2 is a potent pericyte-stimulating factor in tumors. Mechanistically, FGF-2 binds to FGFR2 to stimulate pericyte proliferation and orchestrates the PDGFRβ signaling for vascular recruitment. FGF-2 sensitizes the PDGFRβ signaling through increasing PDGFRβ levels in pericytes. To ensure activation of PDGFRβ, the FGF-2–FGFR1-siganling induces PDGF-BB and PDGF-DD, two ligands for PDGFRβ, in angiogenic endothelial cells. Thus, FGF-2 directly and indirectly stimulates pericyte proliferation and recruitment by modulating the PDGF–PDGFRβ signaling. Our study identifies a novel mechanism by which the FGF-2 and PDGF-BB collaboratively modulate perivascular cell coverage in tumor vessels, thus providing mechanistic insights of pericyte–endothelial cell interactions in TME and conceptual implications for treatment of cancers and other diseases by targeting the FGF-2–FGFR-pericyte axis.

Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy.

Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.

Abstract 3090: LY3207447, a tetravalent bispecific antibody targeting VEGFR2 and angiopoietin-2, provides a more efficient anti-angiogenic therapy and an alternative for combination

Abstract Angiopoietin-2 (Ang-2) is released from endothelial cells only in response to stimulus (e.g. wound healing, tumor growth) and facilitates blood vessel sprouting and inhibits pericyte-endothelial cell interaction via Tie2 signaling. In tumors, Ang-2 is up-regulated and acts together with the VEGF/VEGFR2 pathway to stimulate tumor angiogenesis and metastasis. While therapeutic intervention using antagonists to the VEGF/VEGFR2 pathway has proven to be successful in limiting disease progression in a number of different clinical settings, there is an obvious need for an improved response. In various preclinical mouse angiogenesis or xenograft models, the combination treatment with anti-Ang2 antibody and the VEGF/VEGFR blocker provided additional benefit over inhibiting the individual pathway. Here as an alternative to combo therapy, we have engineered a tetravalent IgG-scFv bispecific antibody, LY3207447. LY3207447 is an immunoglobulin G4 (IgG4) antibody, comprising of VEGFR2 antibody derived from ramucirumab and a C-terminally fused single-chain variable fragment (scFv) targeting Ang2. LY3207447 binds to both the extracellular domain of VEGFR2 and soluble Ang2 with high affinity and blocks binding of Ang2 to Tie2 and VEGF to VEGFR2, and therefore inhibits signaling. We have shown that LY3207447 blocks binding of human Ang-2 to human Tie2-Fc by an ELISA assay and neutralizes Ang-2 induced phospho-Tie-2, but not Ang-1 induced phospho-Tie-2 in CHO cells overexpressing Tie-2 receptor. Moreover, LY3207447 neutralizes human VEGF165-induced phospho-VEGFR2 stimulation, cord formation and cell proliferation in human endothelial colony forming cells (ECFCs) and human dermal microvascular endothelial cells (HMVEC-d). LY3207447 blocks human and cyno VEGFR2, but not rodent VEGFR2. For anti-Ang2 arm, LY3207447 blocks human, cyno and mouse Ang2. Pre-clinical evaluation of LY3207447 in mouse retinal angiogenesis model resulted in an abrogation of angiogenesis. Combination studies using a rodent-specific surrogate VEGFR2 blocking antibody, DC101, with parental Ang2 antibody from which the scFv was derived from, inhibited both tumor growth and metastasis, resulting in increased survival compared to monotherapies in mouse xenograft model. These data establish VEGFR2/Ang2 bispecific antibodies as a promising anti-angiogenic, anti-metastatic and anti-tumor agent for the treatment of cancer in combination with other therapies. Citation Format: Jonathan Tetreault, Sudhakar Chintharlapalli, Donmienne Leung, Damien Gerald, Linda Lee, Rowena Almonte-Baldonado, Lysiane Huber, Jianghuai Xu, Bharathi Ramamurthy, Jennifer Pereira, Johnny Croy, Jirong Lu, Ling Liu. LY3207447, a tetravalent bispecific antibody targeting VEGFR2 and angiopoietin-2, provides a more efficient anti-angiogenic therapy and an alternative for combination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3090. doi:10.1158/1538-7445.AM2017-3090

Abstract 3259: LY3127804, a novel anti-Angiopoietin-2 antibody in combination with an anti-VEGFR2 antibody potently inhibits angiogenesis, tumor growth and metastasis

Abstract Angiopoeitin-2 (Ang2) is released from endothelial cells only in response to stimulus (e.g. wound healing, tumor growth) and facilitates blood vessel sprouting and inhibits pericyte-endothelial cell interaction via Tie2 signaling. Combination of an anti-Ang2 antibody and aflibercept, a VEGF trap, has been shown to inhibit tumor growth and decrease tumor vascularity in mouse xenograft tumor models (Daly et al., Cancer Res (2013) 73(1):108). Multiple investigational anti-Ang2 antibody therapies are currently in clinical trials. LY3127804 is a humanized and engineered IgG4 isotype antibody that selectively binds to Ang2 with high affinity and neutralizes Ang2 induced phospho-Tie2. LY3127804 inhibits sprouting angiogenesis and increases pericyte coverage in a mouse developmental retinal angiogenesis model and in mice bearing PC3 xenograft tumors. Combination of LY3127804 and DC101, a potent anti-VEGFR2 antibody, exhibits enhanced efficacy when compared to monotherapy in multiple patient derived xenograft models including NSCLC and ovarian cancers. Anti-Ang2 antibody monotherapy alone resulted in marginal reduction of tumor growth and improved overall survival, while DC101monotherapy had greater reduction in tumor volume with no survival benefit in MDA-MB-231 breast orthotopic model. Combination of anti-Ang2 antibody with anti-VEGFR2 antibody shows reduction in tumor volume and improved overall survival. This robust pre-clinical evidence supports testing the combination of anti-Ang2 and anti-VEGFR2 antibodies in the clinic. LY3127804 is currently in Phase 1 clinical trials (NCT02597036) Citation Format: Sudhakar R. Chintharlapalli, Johnny E. Croy, Donmienne Leung, Damien Gerald, Jirong Lu, Philip W. Iversen, Linda N. Lee, Lysiane Huber, Jonathan Tetreault, Rowena Almonte-Baldonado, Jianghuai Xu, Bharathi Ramamurthy, Jennifer A. Pereira, Chi-Kin Chow, Axel-Rainer Hanauske, Volker Wacheck, Laura Benjamin, Ling Liu. LY3127804, a novel anti-Angiopoietin-2 antibody in combination with an anti-VEGFR2 antibody potently inhibits angiogenesis, tumor growth and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3259.

Two- and three-dimensional co-culture models of soft tissue healing: pericyte-endothelial cell interaction.

The demographic change in western countries towards an older population is being shadowed by an increased appearance of chronic diseases influencing soft tissue healing in a negative manner. Although various promising therapeutic approaches are available for treating chronic wounds, no in vitro model exists that successfully allows the analysis of interacting cells and of the effect of therapeutic drugs within a wound. Granulation tissue assures wound stability, neo-angiogenesis and revascularization finally leading to functional soft tissue repair. As one of the first steps in developing a model for human granulation tissue, we examined microvascular endothelial cells and pericytes in conventional 2D and in 3D spheroid co-cultures. We determined which parameters could be used in a standardized manner and whether the cultures were responsive to hypoxia and to erythropoietin supplementation. The read-out parameters of cell migration, cell density, rate of apoptotic cells, spatial cell distribution in the spheroid and spheroid volume were shown to be excellent analytic measures. In addition, quantification of hypoxia-related genes identified a total of 13 genes that were up-regulated in spheroids after hypoxia. As these parameters delivered reliable results in the present approach and as the general morphological distribution of pericytes and endothelial cells within the spheroid occurred in a typical manner, we believe that this basic in vitro model will serve for the future study of diverse aspects of soft tissue healing.

A novel angiogenic peptide, ΔADT: A truncated adrenotensin peptide revealed by secretory peptidome analysis of human retinal pericytes.

Retinal pericytes play an important role in the maintenance of retinal microvascular homeostasis. We performed a secretory peptidome of primary human retinal pericytes. Using liquid chromatography-tandem mass spectrometry analysis in the culture medium of retinal pericytes, we identified 256 peptides derived from 114 proteins, and identified a novel partial fragment Leu163-His183 (termed ΔADT) of adrenotensin (ADT). To elucidate the role of ΔADT as a soluble mediator of pericyte-endothelial cell interactions, we investigated the bioactivity of ΔADT in human retinal microvascular endothelial cells (HRMVECs). The cell proliferation assay indicated that the proliferation of HRMVECs was promoted by ADT or ΔADT. Moreover, ΔADT had a greater growth promoting effect than ADT in HRMVECs and induced migration and tube formation of HRMVECs. We also observed actin reorganization and that the levels of phosphorylated focal adhesion kinase in ΔADT stimulated HRMVECs. These results showed that ΔADT induces profound actin reorganization and increases the levels of phosphorylated focal adhesion kinase. Collectively, our study showed that ΔADT has an angiogenic activity, and suggested that ΔADT is a novel angiogenic peptide.

An ex vivo model for anti-angiogenic drug testing on intact microvascular networks.

New models of angiogenesis that mimic the complexity of real microvascular networks are needed. Recently, our laboratory demonstrated that cultured rat mesentery tissues contain viable microvascular networks and could be used to probe pericyte-endothelial cell interactions. The objective of this study was to demonstrate the efficacy of the rat mesentery culture model for anti-angiogenic drug testing by time-lapse quantification of network growth. Mesenteric windows were harvested from adult rats, secured in place with an insert, and cultured for 3 days according to 3 experimental groups: 1) 10% serum (angiogenesis control), 2) 10% serum + sunitinib (SU11248), and 3) 10% serum + bevacizumab. Labeling with FITC conjugated BSI-lectin on Day 0 and 3 identified endothelial cells along blood and lymphatic microvascular networks. Comparison between day 0 (before) and 3 (after) in networks stimulated by 10% serum demonstrated a dramatic increase in vascular density and capillary sprouting. Growing networks contained proliferating endothelial cells and NG2+ vascular pericytes. Media supplementation with sunitinib (SU11248) or bevacizumab both inhibited the network angiogenic responses. The comparison of the same networks before and after treatment enabled the identification of tissue specific responses. Our results establish, for the first time, the ability to evaluate an anti-angiogenic drug based on time-lapse imaging on an intact microvascular network in an ex vivo scenario.

NG2 proteoglycan-dependent recruitment of tumor macrophages promotes pericyte-endothelial cell interactions required for brain tumor vascularization

Early stage growth of intracranial B16F10 tumors is reduced by 87% in myeloid-specific NG2 null (Mac-NG2ko) mice and by 77% in pericyte-specific NG2 null (PC-NG2ko) mice, demonstrating the importance of the NG2 proteoglycan in each of these stromal compartments. In both genotypes, loss of pericyte-endothelial cell interaction results in numerous structural defects in tumor blood vessels, including decreased formation of endothelial cell junctions and decreased assembly of the vascular basal lamina. All vascular deficits are larger in Mac-NG2ko mice than in PC-NG2ko mice, correlating with the greater decrease in pericyte-endothelial cell interaction in Mac-NG2ko animals. Accordingly, tumor vessels in Mac-NG2ko mice have a smaller diameter, lower degree of patency, and higher degree of leakiness than tumor vessels in PC-NG2ko mice, leading to less efficient tumor blood flow and to increased intratumoral hypoxia. While reduced pericyte interaction with endothelial cells in PC-NG2ko mice is caused by loss of NG2-dependent pericyte activation of β1 integrin signaling in endothelial cells, reduced pericyte-endothelial cell interaction in Mac-NG2ko mice is due to a 90% reduction in NG2-dependent macrophage recruitment to tumors. The absence of a macrophage-derived signal(s) in Mac-NG2ko mice results in the loss of pericyte ability to associate with endothelial cells, possibly due to reduced expression of N-cadherin by both pericytes and endothelial cells.

Emerging roles of pericytes in the regulation of the neurovascular unit in health and disease.

Pericytes of the central nervous system (CNS) are uniquely positioned within a multicellular structure termed the neurovascular unit (NVU) to provide crucial support to blood brain barrier (BBB) formation, maintenance, and stability. Numerous CNS diseases are associated with some aspect of BBB dysfunction. A dysfunction can manifest as one or multiple disruptions to any of the following barriers: physical, metabolic, immunological and transport barrier. A breach in the BBB can notably result in BBB hyper-permeability, endothelial activation and enhanced immune-endothelial interaction. How the BBB is regulated within this integrated unit remains largely unknown, especially as it relates to pericyte-endothelial interaction. We summarize the latest findings on pericyte origin, possible marker expression, and availability within different organ systems. We highlight pericyte-endothelial cell interactions, concentrating on extra- and intra- cellular signaling mechanisms linked to platelet derived growth factor-B, transforming growth factor -β, angiopoietins, Notch, and gap junctions. We discuss the role of pericytes in the NVU under inflammatory insult, focusing on how pericytes may indirectly affect leukocyte CNS infiltration, the direct role of pericyte-mediated basement membrane modifications, and immune responses. We review new findings of pericyte actions in CNS pathologies including Alzheimer's disease, stroke, multiple sclerosis, diabetic retinopathy, and HIV-1 infection. The uncovering of the regulatory role of pericytes on the BBB will provide key insight into how barrier integrity can be re-established during neuroinflammation.

Purification and culture of central nervous system pericytes.

Pericytes are found on the abluminal surface of endothelial tubes. The function of these cells is still being elucidated, but they have been shown to be important for development and maintenance of the blood-brain barrier, regulation of angiogenesis and capillary blood flow, and regulation of the neural response to injury and disease. Previously used methods to isolate pericytes have relied on negative selection and prolonged culture of microvessel cells and may lead to populations of pericytes contaminated by other neural cell types. We have developed an immunopanning protocol to specifically purify pericytes from capillaries in the rodent optic nerve. This method relies on a combination of negative and positive selection criteria and allows prospective, acute isolation of pericytes. Use of this method will facilitate studies of pericyte cell biology and function and pericyte-endothelial cell interactions.

Foxc1 is required by pericytes during fetal brain angiogenesis

SummaryBrain pericytes play a critical role in blood vessel stability and blood–brain barrier maturation. Despite this, how brain pericytes function in these different capacities is only beginning to be understood. Here we show that the forkhead transcription factor Foxc1 is expressed by brain pericytes during development and is critical for pericyte regulation of vascular development in the fetal brain. Conditional deletion of Foxc1 from pericytes and vascular smooth muscle cells leads to late-gestation cerebral micro-hemorrhages as well as pericyte and endothelial cell hyperplasia due to increased proliferation of both cell types. Conditional Foxc1 mutants do not have widespread defects in BBB maturation, though focal breakdown of BBB integrity is observed in large, dysplastic vessels. qPCR profiling of brain microvessels isolated from conditional mutants showed alterations in pericyte-expressed proteoglycans while other genes previously implicated in pericyte–endothelial cell interactions were unchanged. Collectively these data point towards an important role for Foxc1 in certain brain pericyte functions (e.g. vessel morphogenesis) but not others (e.g. barriergenesis).