Primary Human Pericytes Research Articles

Evaluation of Barrier Integrity Using a Two-Layered Microfluidic Device Mimicking the Blood-Brain Barrier.

The blood-brain barrier (BBB) plays an essential role in maintaining the homeostasis of the brain microenvironment by controlling the influx and efflux of biological substances that are necessary to sustain the neuronal metabolic activity and functions. This barrier is established at the blood-brain interface of the brain microcapillaries by different cells. These include microvascular endothelial cells, astrocytes, and pericytes besides other components such as microglia, basal membrane, and neuronal cells forming together what is commonly referred to as the neurovascular unit; different in vivo and in vitro platforms are available to study the BBB where each system provides specific benefits and drawbacks. Recently, organ-on-a-chip platforms combine the elegance of microengineering technology with the complexity of biological systems to create near-ideal experimental models for various diseases and organs. These microfluidic devices with micron-sized channels allow the cells to be grown in a more biologically relevant environment, enabling cell to cell communications with continuous bathing in biological fluids in a tissue-like fashion. They also closely represent tissue and organ functionality by recapitulating mechanical forces as well as vascular perfusion. Here, we describe the use of humanized BBB model created with microfluidic organ-on-a-chip technology where human brain microvascular endothelial cells (BMECs) are cocultured with primary human pericytes and astrocytes. We thoroughly described the method to assess BBB integrity using a microfluidic chip and various sizes of labeled dextran as permeability markers. In addition, we provide a detailed protocol on how to microscopically investigate the tight junction proteins expression between hBMECs.

Differential effects of SARS-CoV-2 variants on central nervous system cells and blood–brain barrier functions

BackgroundAlthough mainly causing a respiratory syndrome, numerous neurological symptoms have been identified following of SARS-CoV-2 infection. However, how the virus affects the brain and how the mutations carried by the different variants modulate those neurological symptoms remain unclear.MethodsWe used primary human pericytes, foetal astrocytes, endothelial cells and a microglial cell line to investigate the effect of several SARS-CoV-2 variants of concern or interest on their functional activities. Cells and a 3D blood–brain barrier model were infected with the wild-type form of SARS-CoV-2, Alpha, Beta, Delta, Eta, or Omicron (BA.1) variants at various MOI. Cells and supernatant were used to evaluate cell susceptibility to the virus using a microscopic assay as well as effects of infection on (i) cell metabolic activity using a colorimetric MTS assay; (ii) viral cytopathogenicity using the xCELLigence system; (iii) extracellular glutamate concentration by fluorometric assay; and (iv) modulation of blood–brain barrier permeability.ResultsWe demonstrate that productive infection of brain cells is SARS-CoV-2 variant dependent and that all the variants induce stress to CNS cells. The wild-type virus was cytopathic to all cell types except astrocytes, whilst Alpha and Beta variants were only cytopathic for pericytes, and the Omicron variant cytopathic for endothelial cells and pericytes. Lastly wild-type virus increases blood–brain barrier permeability and all variants, except Beta, modulate extracellular glutamate concentration, which can lead to excitotoxicity or altered neurotransmission.ConclusionsThese results suggest that SARS-CoV-2 is neurotropic, with deleterious consequences for the blood–brain barrier integrity and central nervous system cells, which could underlie neurological disorders following SARS-CoV-2 infection.

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.

New In Vitro Model to Study Multicellular and Flow Control of Blood‐Brain Barrier

Brain endothelial cells of the blood‐brain barrier (BBB) have been shown to be regulated by supportive cells, such as pericytes and astrocytes, and shear stress exposure. However, studies investigating the impact of pericytes and astrocytes on brain endothelial cell function have identified both beneficial and detrimental results. Additionally, most studies investigating the relationship between shear stress and brain endothelial cell function lack physiological relevance via the use of sub‐physiological shear stress magnitudes and/or via the absence of pericytes and astrocytes. In this study, we developed a millifluidic device compatible with standard transwell inserts to investigate BBB function. In contrast to standard polydimethylsiloxane (PDMS) microfluidic devices, this model allows for easy, reproducible shear stress exposure without common limitations of PDMS devices such as inadequate nutrient diffusion and air bubble formation. In no‐flow conditions, we first used the device to examine the impact of primary human pericytes and astrocytes on human brain microvascular endothelial cell (HBMEC) barrier integrity. We found that astrocytes, pericytes, and a 1:1 ratio of both cell types increased HBMEC barrier integrity via reduced permeability to 40 kDa fluorescent dextran, which was associated with increased expression of tight junction protein, claudin‐5. Interestingly, we also observed a significantly lower permeability to 3 kDa dextran in HBMEC‐pericyte co‐cultures compared to HBMEC‐astrocyte and HBMEC‐astrocyte‐pericyte co‐cultures. Based on these findings, we hypothesize pericytes may be providing increased barrier support to the BBB model compared to astrocytes although they both function as permeability reducers. After using the device to generate 24‐hour flow at 12 dynes/cm2, we observed that shear stress exposure significantly reduced dextran permeability in HBMEC monolayers, but not in tri‐culture models consisting of HBMECs, pericytes, and astrocytes. These results indicate that co‐cultures may demonstrate a more pronounced impact on overall BBB permeability than flow exposure. In both cases, flow exposure was interestingly associated with reduced expression of both claudin‐5 and occludin. However, ZO‐1 expression, and localization at cell‐cell junctions increased in the tri‐culture but exhibited no apparent change in the HBMEC monolayer. Under flow conditions, we also observed alignment of HBMECs in the tri‐culture while no such phenomenon was observed in HBMEC monolayers, indicating supportive cells and flow are both essential to observe brain endothelial cell alignment in vitro. Collectively, these results support the necessity of physiologically relevant, multicellular BBB models when investigating brain endothelial cell function in relation to the BBB. Additionally, our findings provide clues on the role of shear stress and supportive cells within the BBB, a critical step to elucidating the physiology of the neurovascular unit.

Pericytes' Circadian Clock Affects Endothelial Cells' Synchronization and Angiogenesis in a 3D Tissue Engineered Scaffold.

Angiogenesis, the formation of new capillaries from existing ones, is a fundamental process in regenerative medicine and tissue engineering. While it is known to be affected by circadian rhythms in vivo, its peripheral regulation within the vasculature and the role it performs in regulating the interplay between vascular cells have not yet been investigated. Peripheral clocks within the vasculature have been described in the endothelium and in smooth muscle cells. However, to date, scarce evidence has been presented regarding pericytes, a perivascular cell population deeply involved in the regulation of angiogenesis and vessel maturation, as well as endothelial function and homeostasis. More crucially, pericytes are also a promising source of cells for cell therapy and tissue engineering. Here, we established that human primary pericytes express key circadian genes and proteins in a rhythmic fashion upon synchronization. Conversely, we did not detect the same patterns in cultured endothelial cells. In line with these results, pericytes’ viability was disproportionately affected by circadian cycle disruption, as compared to endothelial cells. Interestingly, endothelial cells’ rhythm could be induced following exposure to synchronized pericytes in a contact co-culture. We propose that this mechanism could be linked to the altered release/uptake pattern of lactate, a known mediator of cell-cell interaction which was specifically altered in pericytes by the knockout of the key circadian regulator Bmal1. In an angiogenesis assay, the maturation of vessel-like structures was affected only when both endothelial cells and pericytes did not express Bmal1, indicating a compensation system. In a 3D tissue engineering scaffold, a synchronized clock supported a more structured organization of cells around the scaffold pores, and a maturation of vascular structures. Our results demonstrate that pericytes play a critical role in regulating the circadian rhythms in endothelial cells, and that silencing this system disproportionately affects their pro-angiogenic function. Particularly, in the context of tissue engineering and regenerative medicine, considering the effect of circadian rhythms may be critical for the development of mature vascular structures and to obtain the maximal reparative effect.

A Quasi-Physiological Microfluidic Blood-Brain Barrier Model for Brain Permeability Studies.

Microfluidics-based organ-on-a-chip technology allows for developing a new class of in-vitro blood-brain barrier (BBB) models that recapitulate many hemodynamic and architectural features of the brain microvasculature not attainable with conventional two-dimensional platforms. Herein, we describe and validate a novel microfluidic BBB model that closely mimics the one in situ. Induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs) were juxtaposed with primary human pericytes and astrocytes in a co-culture to enable BBB-specific characteristics, such as low paracellular permeability, efflux activity, and osmotic responses. The permeability coefficients of [13C12] sucrose and [13C6] mannitol were assessed using a highly sensitive LC-MS/MS procedure. The resulting BBB displayed continuous tight-junction patterns, low permeability to mannitol and sucrose, and quasi-physiological responses to hyperosmolar opening and p-glycoprotein inhibitor treatment, as demonstrated by decreased BBB integrity and increased permeability of rhodamine 123, respectively. Astrocytes and pericytes on the abluminal side of the vascular channel provided the environmental cues necessary to form a tight barrier and extend the model’s long-term viability for time-course studies. In conclusion, our novel multi-culture microfluidic platform showcased the ability to replicate a quasi-physiological brain microvascular, thus enabling the development of a highly predictive and translationally relevant BBB model.

PERK Is Critical for Alphavirus Nonstructural Protein Translation

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4–5 log10. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.

Identification and functional characterization of circular RNAs in pericytes

Abstract Background Circular RNAs (circRNAs) are generated by back-splicing. They are known to be robustly expressed in a variety of mammalian cell types and organism and have been reported to influence cell biology by acting e.g. as microRNA sponges or regulating host gene expression. Recently, our group reported functionally relevant circRNA expression in endothelial cells. Despite their important role in the cardiovascular system, the expression and function of circRNAs in pericytes is not well studied. Pericytes are perivascular mural cells, important for vessel maturation and endothelial barrier function. Their recruitment towards endothelial cells is mainly meditated by platelet-derived growth factor (PDGF) signaling. However, a more precise understanding of the regulation of pericyte differentiation and survival is necessary. Objective Here, we analyse circRNA expression in pericytes and demonstrate biological relevance of the hypoxia regulated circular RNA PLOD2 (cPLOD2). Methods and results Using RNA Sequencing in ribosomal depleted RNA we characterized the expression of circRNAs in human pericytes under normoxic and hypoxic (1% O2, 48h) conditions. We identified several circular RNAs being regulated upon hypoxia. The identified circular RNAs demonstrated resistance towards RNase-R digestion and lacking of poly-adenylation. Some of them were found to be localized and in the cytosol, whereas others also occur in the nucleus of the cells. Especially cPLOD2 raised our attention since it is significantly upregulated and robustly expressed upon hypoxia. Silencing cPLOD2 by siRNA resulted in significant de-differentiation of pericytes that went along with a loss of cell viability. Mechanistically, transcription factor screening assays revealed that silencing of cPLOD2 enhances the activity of the transcription factors ELK1/SRF, which have been documented to result in de-differentiation of smooth muscle cells. Conclusion Here we characterize the expression pattern of circRNAs in human primary pericytes. Among others, cPLOD2 significantly regulates pericyte function. Our results indicate hypoxia as a major regulator of circRNA expression in pericytes and show that circRNAs are capable of regulating pericyte function by modulating activity of transcription factors. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Deutsche Forschungsgesellschaft (DFG) - SFB834; Deutsche Gesellschaft für Herz-Kreislaufforschung (DZHK)

Protein Kinase CK2 Regulates Nerve/Glial Antigen (NG)2-Mediated Angiogenic Activity of Human Pericytes.

Protein kinase CK2 is a crucial regulator of endothelial cell proliferation, migration and sprouting during angiogenesis. However, it is still unknown whether this kinase additionally affects the angiogenic activity of other vessel-associated cells. In this study, we investigated the effect of CK2 inhibition on primary human pericytes. We found that CK2 inhibition reduces the expression of nerve/glial antigen (NG)2, a crucial factor which is involved in angiogenic processes. Reporter gene assays revealed a 114 bp transcriptional active region of the human NG2 promoter, whose activity was decreased after CK2 inhibition. Functional analyses demonstrated that the pharmacological inhibition of CK2 by CX-4945 suppresses pericyte proliferation, migration, spheroid sprouting and the stabilization of endothelial tubes. Moreover, aortic rings of NG2−/− mice showed a significantly reduced vascular sprouting when compared to rings of NG2+/+ mice, indicating that NG2 is an important regulator of the angiogenic activity of pericytes. In vivo, implanted Matrigel plugs containing CX-4945-treated pericytes exhibited a lower microvessel density when compared to controls. These findings demonstrate that CK2 regulates the angiogenic activity of pericytes through NG2 gene expression. Hence, the inhibition of CK2 represents a promising anti-angiogenic strategy, because it does not only target endothelial cells, but also vessel-associated pericytes.

Characterization of p38 Inflammatory Response in Human Pericytes

G protein‐coupled receptor (GPCR) mediated mitogen activated protein kinase (MAPK) p38 activation is a crucial regulator of pro‐inflammation and pro‐angiogenic responses during chronic lung disease. Our studies have revealed an atypical (non‐canonical) pathway for p38 activation that induces auto‐phosphorylation of p38 through the direct binding of transforming growth factor β activated kinase 1 binding protein‐1 (TAB1) in endothelial cells. However, during angiogenesis, endothelial cells are known for their rapid reproduction, migration, and tubule formation. Pericyte cells serve to guide these endothelial cells, providing vascular stability and cytokine signaling. During inflammation, pericytes migrate away from the vasculature leaving endothelial tubules exposed and fragile. Atypical p38 activation has not yet been examined in pericyte cells, but is hypothesized to play a key role in regulating G protein‐coupled receptor‐mediated control of vascular function. Very little is known about the cellular mechanisms that control atypical p38 activation, representing a novel therapeutic target. Our preliminary data shows that thrombin and histamine can activate p38 via the atypical p38 signaling pathway in primary human pericytes. As the role of GPCR induced p38 signaling in pericytes has not been examined, our current studies focus on defining this role, including establishing a co‐cultured model of pericyte and endothelial cells. Using this model, we are the first to define the role of atypical p38 signaling in pericytes, along with their vascular formation and function. These studies represent a novel approach to regulate vascular p38 activity, pro‐inflammatory signaling, and chronic lung disease, and this model will provide us with the tools necessary to identify the role of p38 induced inflammation in the migration of pericyte cells.

The amyloid-\u03b2 degradation intermediate A\u03b234 is pericyte-associated and reduced in brain capillaries of patients with Alzheimer\u2019s disease

An impairment of amyloid β-peptide (Aβ) clearance is suggested to play a key role in the pathogenesis of sporadic Alzheimer’s disease (AD). Amyloid degradation is mediated by various mechanisms including fragmentation by enzymes like neprilysin, matrix metalloproteinases (MMPs) and a recently identified amyloidolytic activity of β-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 cleavage of Aβ40 and Aβ42 results in the formation of a common Aβ34 intermediate which was found elevated in cerebrospinal fluid levels of patients at the earliest disease stages. To further investigate the role of Aβ34 as a marker for amyloid clearance in AD, we performed a systematic and comprehensive analysis of Aβ34 immunoreactivity in hippocampal and cortical post-mortem brain tissue from AD patients and non-demented elderly individuals. In early Braak stages, Aβ34 was predominantly detectable in a subset of brain capillaries associated with pericytes, while in later disease stages, in clinically diagnosed AD, this pericyte-associated Aβ34 immunoreactivity was largely lost. Aβ34 was also detected in isolated human cortical microvessels associated with brain pericytes and its levels correlated with Aβ40, but not with Aβ42 levels. Moreover, a significantly decreased Aβ34/Aβ40 ratio was observed in microvessels from AD patients in comparison to non-demented controls suggesting a reduced proteolytic degradation of Aβ40 to Aβ34 in AD. In line with the hypothesis that pericytes at the neurovascular unit are major producers of Aβ34, biochemical studies in cultured human primary pericytes revealed a time and dose dependent increase of Aβ34 levels upon treatment with recombinant Aβ40 peptides while Aβ34 production was impaired when Aβ40 uptake was reduced or BACE1 activity was inhibited. Collectively, our findings indicate that Aβ34 is generated by a novel BACE1-mediated Aβ clearance pathway in pericytes of brain capillaries. As amyloid clearance is significantly reduced in AD, impairment of this pathway might be a major driver of the pathogenesis in sporadic AD.

Human primary endothelial cells are impaired in nucleotide excision repair and sensitive to benzo[a]pyrene compared with smooth muscle cells and pericytes

The endothelium represents the inner cell layer of blood vessels and is supported by smooth muscle cells and pericytes, which form the vessel structure. The endothelium is involved in the pathogenesis of many diseases, including the development of atherosclerosis. Due to direct blood contact, the blood vessel endothelium is inevitably exposed to genotoxic substances that are systemically taken up by the body, including benzo[a]pyrene, which is a major genotoxic component in cigarette smoke and a common environmental mutagen and human carcinogen. Here, we evaluated the impact of benzo[a]pyrene diol epoxide (BPDE), which is the reactive metabolite of benzo[a]pyrene, on the three innermost vessel cell types. Primary human endothelial cells (HUVEC), primary human smooth muscle cells (HUASMC) and primary human pericytes (HPC) were treated with BPDE, and analyses of cytotoxicity, cellular senescence and genotoxic effects were then performed. The results showed that HUVEC were more sensitive to the cytotoxic activity of BPDE than HUASMC and HPC. We further show that HUVEC display a detraction in the repair of BPDE-induced adducts, as determined through the comet assay and the quantification of BPDE adducts in post-labelling experiments. A screening for DNA repair factors revealed that the nucleotide excision repair (NER) proteins ERCC1, XPF and ligase I were expressed at lower levels in HUVEC compared with HUASMC and HPC, which corresponds with the impaired NER-mediated removal of BPDE adducts from DNA. Taken together, the data revealed that HUVEC exhibit an unexpected DNA repair-impaired phenotype, which has implications on the response of the endothelium to genotoxicants that induce bulky DNA lesions, including the development of vascular diseases resulting from smoking and environmental pollution.

Quantitative Label-Free Imaging of 3D Vascular Networks Self-Assembled in Synthetic Hydrogels.

Vascularization is an important strategy to overcome diffusion limits and enable the formation of complex, physiologically relevant engineered tissues and organoids. Self-assembly is a technique to generate in vitro vascular networks, but engineering the necessary network morphology and function remains challenging. Here, autofluorescence multiphoton microscopy (aMPM), a label-free imaging technique, is used to quantitatively evaluate in vitro vascular network morphology. Vascular networks are generated using human embryonic stem cell-derived endothelial cells and primary human pericytes encapsulated in synthetic poly(ethylene glycol)-based hydrogels. Two custom-built bioreactors are used to generate distinct fluid flow patterns during vascular network formation: recirculating flow or continuous flow. aMPM is used to image these 3D vascular networks without the need for fixation, labels, or dyes. Image processing and analysis algorithms are developed to extract quantitative morphological parameters from these label-free images. It is observed with aMPM that both bioreactors promote formation of vascular networks with lower network anisotropy compared to static conditions, and the continuous flow bioreactor induces more branch points compared to static conditions. Importantly, these results agree with trends observed with immunocytochemistry. These studies demonstrate that aMPM allows label-free monitoring of vascular network morphology to streamline optimization of growth conditions and provide quality control of engineered tissues.

Nitric Oxide Mediates Downregulation of Tissue Factor Expression in Primary Human Pericytes through a p38 MAPK Signaling Pathway

Tissue factor (TF) is a high-affinity receptor for FVII/FVIIa that serves as a key initiator of hemostasis and is thought to also play a functional role in angiogenesis. Elevated TF expression has been linked to upregulated angiogenesis in malignant tumors, while reducing TF expression in experimental tumor models results in decreased angiogenesis. Although these data suggest that high TF expression is critical for angiogenesis, we have reported that TF expression declines significantly in pericytes that surround angiogenic vessels at sites of wound healing. This is the only known example of active TF downregulation, suggesting that pericytes regulate their expression of TF by a unique mechanism. Additionally, TF expression increases in response to many mediators, yet none that decrease TF expression have been described. The goal of this study was to characterize TF downregulation in pericytes and identify mediators of this process.We have previously shown that TF expression in primary cultures of human pericytes is not affected by treatment with various growth factors or inflammatory stimuli, but decreases significantly in response to phorbol 12-myristate 13-acetate (PMA). PMA triggers degradation of TF protein and inhibition of TF mRNA synthesis, both in a Protein Kinase C (PKC)- dependent manner. To identify other signaling molecules in this pathway we used chemical inhibitors to block the activity of signaling molecules downstream of PKC before adding PMA to pericyte cultures. Inhibition of NF-kB, ERK1/2, AKT, JNK, and p38 MAPK did not block degradation of TF protein. However, pericytes that received a p38 inhibitor (SB202190) alone demonstrated significant reduction of TF mRNA. Treatment with SB202190 followed by PMA produced an additive effect on TF mRNA reduction. Western blotting showed that prolonged PMA treatment (>4 hours) produced a sustained decrease in p38 phosphorylation. These data suggest that PMA inhibits p38 activity, and that p38 confers stability to TF mRNA.We have previously found that basic Fibroblast Growth Factor (bFGF) triggers downregulation of pericyte TF a co-culture system with human microvascular endothelial cells. However, transferring bFGF-conditioned endothelial cell media to pericytes cultured alone failed to reproduce TF loss. bFGF has been shown to stimulate nitric oxide (NO) production, and both bFGF and NO have been linked to angiogenesis. This led us to consider NO as a potential labile mediator of TF downregulation. ±6.1%, p<0.01). However, expression of TF mRNA was not reduced at this time, as it is during culture with PMA. Pericytes treated with DETA NO demonstrated sustained p38 phosphorylation for up to 8 hours. Taken together, these data suggest that DETA NO downregulates TF protein but maintains basal levels of TF mRNA, potentially in a p38-dependent manner.Based on these data, we hypothesize that the p38 signaling axis is a key component of a unique pathway of TF regulation in pericytes, and that endothelial nitric oxide contributes to downregulation of pericyte TFin vivo at sites of physiologic angiogenesis. DisclosuresHoffman:Novo Nordisk A/S: Consultancy, Honoraria, Research Funding.

Identification and Functional Characterization of Hypoxia-Induced Endoplasmic Reticulum Stress Regulating lncRNA (HypERlnc) in Pericytes.

Pericytes are essential for vessel maturation and endothelial barrier function. Long noncoding RNAs regulate many cellular functions, but their role in pericyte biology remains unexplored. Here, we investigate the effect of hypoxia-induced endoplasmic reticulum stress regulating long noncoding RNAs (HypERlnc, also known as ENSG00000262454) on pericyte function in vitro and its regulation in human heart failure and idiopathic pulmonary arterial hypertension. RNA sequencing in human primary pericytes identified hypoxia-regulated long noncoding RNAs, including HypERlnc. Silencing of HypERlnc decreased cell viability and proliferation and resulted in pericyte dedifferentiation, which went along with increased endothelial permeability in cocultures consisting of human primary pericyte and human coronary microvascular endothelial cells. Consistently, Cas9-based transcriptional activation of HypERlnc was associated with increased expression of pericyte marker genes. Moreover, HypERlnc knockdown reduced endothelial-pericyte recruitment in Matrigel assays (P<0.05). Mechanistically, transcription factor reporter arrays demonstrated that endoplasmic reticulum stress-related transcription factors were prominently activated by HypERlnc knockdown, which was confirmed via immunoblotting for the endoplasmic reticulum stress markers IRE1α (P<0.001), ATF6 (P<0.01), and soluble BiP (P<0.001). Kyoto encyclopedia of genes and gene ontology pathway analyses of RNA sequencing experiments after HypERlnc knockdown indicate a role in cardiovascular disease states. Indeed, HypERlnc expression was significantly reduced in human cardiac tissue from patients with heart failure (P<0.05; n=19) compared with controls. In addition, HypERlnc expression significantly correlated with pericyte markers in human lungs derived from patients diagnosed with idiopathic pulmonary arterial hypertension and from donor lungs (n=14). Here, we show that HypERlnc regulates human pericyte function and the endoplasmic reticulum stress response. In addition, RNA sequencing analyses in conjunction with reduced expression of HypERlnc in heart failure and correlation with pericyte markers in idiopathic pulmonary arterial hypertension indicate a role of HypERlnc in human cardiopulmonary disease.

Downregulation of Tissue Factor in Pericytes By a Protein Kinase C-Dependent Mechanism

Tissue factor (TF) is a transmembrane protein in the type II cytokine receptor family that serves as a cofactor for factor VIIa activity, and is essential for normal initiation of hemostasis. Its expression is generally upregulated in the settings of inflammation, immune activation, tissue injury and malignancy. TF has biological functions in addition to its role hemostasis. In malignancy, high expression of TF by tumor cells promotes local angiogenesis. By contrast, we have reported that expression of perivascular TF is downregulated within 24 hours after wounding, and remains low around angiogenic vessels during physiologic wound healing. The mechanism(s) of physiologic downregulation of TF, and its role in physiologic angiogenesis have not been previously characterized. Therefore, the objective of this study was to identify mechanisms contributing to TF downregulation in primary human pericytes. We determined that loss of TF in human pericytes can be recapitulated in vitro by treating pericyte cultures with phorbol 12-myristate 13-acetate (PMA). The time course and degree of TF downregulation over 24 hours, as shown in the figure, is similar to that observed within the first 24 hours in vivo. Since PMA is a potent activator of Protein Kinase C (PKC), we determined the role of PKC in PMA-mediated TF loss by pretreating pericytes with the broad spectrum PKC inhibitor, Go 6983, prior to stimulation with PMA. PKC inhibition eliminated PMA-induced TF loss, supporting the conclusion that the PMA effect occurs through a PKC-dependent mechanism. PKCa has been shown to induce phosphorylation of the TF cytoplasmic tail, which results in budding of TF-rich microvesicles from the plasma membrane. PMA also triggers activation of the cell-surface protease ADAM17. Based on domain homology with other targets, ADAM17 could potentially cleave and release the extracellular domain of TF from the cell surface. To determine if TF loss occurs through its release from the cell by either mechanism we utilized biotin labeling of surface TF, streptavidin pull down, and western blot analysis to assess trafficking of TF into the culture media. Our results show that surface TF significantly decreases within 2 hours of PMA treatment. However, we failed to detect biotinylated TF in the culture media. Isolation of microvesicles from the media followed by western blotting for TF showed that pericytes do release TF-associated microvesicles at a basal rate, but this rate is not affected by PMA. Taken together, these data show that PMA induces downregulation of TF in primary human pericytes through a PKC-dependent mechanism, and that this mechanism does not stimulate release of TF from the cell in either microvesicles or by proteolytic cleavage of the TF extracellular domain. Our working hypothesis is that PMA triggers internalization of surface TF via activation of protein kinase C, leading to its subsequent degradation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pericyte NF-κB activation enhances endothelial cell proliferation and proangiogenic cytokine secretion in vitro.

Pericytes are skeletal muscle resident, multipotent stem cells that are localized to the microvasculature. In vivo, studies have shown that they respond to damage through activation of nuclear-factor kappa-B (NF-κB), but the downstream effects of NF-κB activation on endothelial cell proliferation and cell–cell signaling during repair remain unknown. The purpose of this study was to examine pericyte NF-κB activation in a model of skeletal muscle damage; and use genetic manipulation to study the effects of changes in pericyte NF-κB activation on endothelial cell proliferation and cytokine secretion. We utilized scratch injury to C2C12 cells in coculture with human primary pericytes to assess NF-κB activation and monocyte chemoattractant protein-1 (MCP-1) secretion from pericytes and C2C12 cells. We also cocultured endothelial cells with pericytes that expressed genetically altered NF-κB activation levels, and then quantified endothelial cell proliferation and screened the conditioned media for secreted cytokines. Pericytes trended toward greater NF-κB activation in injured compared to control cocultures (P = 0.085) and in comparison to C2C12 cells (P = 0.079). Second, increased NF-κB activation in pericytes enhanced the proliferation of cocultured endothelial cells (1.3-fold, P = 0.002). Finally, we identified inflammatory signaling molecules, including MCP-1 and interleukin 8 (IL-8) that may mediate the crosstalk between pericytes and endothelial cells. The results of this study show that pericyte NF-κB activation may be an important mechanism in skeletal muscle repair with implications for the development of therapies for musculoskeletal and vascular diseases, including peripheral artery disease.

Shear Stress Induces Vasoprotective Gene Upregulation in Pericytes

Introduction: Fibrosis is an initial step in the development of atherosclerotic lesions, hallmarked by myofibroblasts entering the tunica media and forming a fibrous cap. Vasa vasorum in the adventitia consist of endothelial cells, surrounded by pericytes. Pericytes can be regarded as cells with stem cell like properties and the capacity to differentiate into myofibroblasts. Although it is known that shear stress induces atherosclerotic lesions, nothing is known about its impact on pericytes. Herein, we investigate the effect of shear stress on pericytes and focus on differentiation into myofibroblasts. Methods: Primary human pericytes (PCs) were isolated from donor fatty tissue and cultured in DMEM + 10% FCS. Primary cells were characterised by immunofluorescent (IF) staining (NG2, CD90, CD105, CD44, CD45, CD73, CD146, CD31, and PDGFR-beta). Endothelial cells (HUVEC) or PC were seeded into flow chambers and subjected to laminar flow at low 10 dyn, high 30 dyn and no shear stress rates for 48 h (n = 3/group). RNA was extracted and analysed by qPCR for tissue inhibitor of metalloproteinase 3, versican and genes known to be regulated in pericyte – myofibroblast transition. In addition, IF staining for cytoskeletal f-actin and VE-cadherin in HUVECS was performed. Results: Characterisation of PCs showed positivity for CD44, Cd90, PDGFR-beta, CD105, NG2, and negativity for CD31, CD45, CD146 and CD73. HUVEC subjected to low and high shear stress aligned and elongated in flow direction. PCs subjected to shear stress, revealed an opposite behavior to HUVEC, aligning almost perpendicular to flow. qPCR analysis of PCs and HUVEC with no, 10 or 30 dyn shear stress revealed that endothelial cells upregulated the extracellular matrix protein versican (2 fold increase of normalized gene expression to GAPDH) and its protease ADAMTS1 (4 fold increase), while TIMP3, a tissue inhibitor of matrix metalloproteinase was upregulated under high shear stress in pericytes (3 fold upregulation). In addition, plasma was upregulated in HUVEC but not PC. Conclusion: Shear stress induces extracellular matrix turnover and in pericytes leads to upregulation of proteases known to stabilise the vascular wall. Pericytes in contrast to endothelial cells align perpendicular to flow direction. Co-culture experiments with pericytes and ECs under flow are planned to verify monoculture results.

An in vitro injury model to examine pericyte‐skeletal muscle cell cross talk

This study aimed to evaluate an in vitro model of cross talk between pericytes and skeletal muscle cells following scrape injury. Nuclear factor‐kappa B (NF‐κB) and monocyte chemoattractant protein‐1 (MCP‐1) are important in the in vivo response to muscle stress and were chosen to evaluate the model. C2C12 cells were co‐cultured with human primary pericytes (HPPs) in transwell inserts. Nuclear NF‐κB/p65 DNA binding activity and MCP‐1 concentration were quantified via ELISA at baseline (BSLN), 3h, 6h, and 24h following scrape injury (INJ) to C2C12 cells. In C2C12 cells, p65 DNA binding activity was significantly elevated at 3h (2.5 fold, p=0.027) and 24h (3.57 fold, p=0.001), relative to BSLN, with no effect of INJ (p=0.698) relative to CON. In HPPs, p65 DNA binding activity was increased relative to BSLN at 6h (2.0 fold, p=0.007), and 24h (2.33 fold, p=0.001). HPPs trended towards greater p65 DNA binding activity in INJ compared to CON (p=0.085) and in comparison to C2C12 cells (p=0.079). At 24h, HPP MCP‐1 secretion was first detected and exceeded C2C12 MCP‐1 secretion (2.1 fold, p&lt;0.001). These data reveal 1) this model is useful for evaluating secreted proteins involved in pericyte‐skeletal muscle cross talk, which are mostly unknown at this time, and 2) novel information regarding HPP MCP‐1 secretion in response to C2C12 scrape injury. This research was supported by an ACSM Foundation Doctoral Student Research Grant.