Pericyte Viability Research Articles

Role of Hippo/ACSL4 Axis in Ferroptosis-Induced Pericyte Loss and Vascular Dysfunction in Sepsis

Sepsis is a critical condition characterized by a systemic inflammatory response to infection, often leading to severe vascular dysfunction and high mortality. One of the hallmarks of vascular dysfunction in sepsis is increased vascular permeability and the loss of pericytes, which are essential for maintaining vascular integrity. Despite the significance of pericyte loss in sepsis, the primary type of cell death responsible and the underlying molecular mechanisms remain incompletely understood. This study aims to elucidate these mechanisms by focusing on ferroptosis, a form of programmed cell death, and its regulation through the Hippo/ACSL4 axis. Our research confirmed significant pericyte loss in patients with sepsis. Through advanced single-cell analysis and proteomics, ferroptosis was identified as a key differentiating cell death type between sepsis and sham samples. Further metabolomics analysis revealed that Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) plays a pivotal role in the ferroptosis of pericytes during sepsis. In vitro experiments demonstrated that downregulation of ACSL4 effectively reduced lipopolysaccharide (LPS)-induced lipid peroxidation, restored pericyte viability, and improved endothelial permeability. In vivo studies with pericyte-specific ACSL4 knockout mice showed a marked decrease in pericyte loss and enhanced vascular barrier function following sepsis induction. To translate these findings into potential therapeutic strategies, we developed pericyte-targeting liposomes encapsulating ACSL4 shRNA adenovirus. These liposomes successfully restored pulmonary vascular barrier function and significantly reduced pericyte loss in septic conditions. The results of this study underscore the crucial role of ACSL4 in mediating ferroptosis in pericytes and highlight the therapeutic potential of targeting ACSL4 to mitigate vascular dysfunction in sepsis.

Rapamycin Treatment Reduces Brain Pericyte Constriction in Ischemic Stroke.

The contraction and subsequent death of brain pericytes may play a role in microvascular no-reflow following the reopening of an occluded artery during ischemic stroke. Mammalian target of rapamycin (mTOR) inhibition has been shown to reduce motility/contractility of various cancer cell lines and reduce neuronal cell death in stroke. However, the effects of mTOR inhibition on brain pericyte contraction and death during ischemia have not yet been investigated. Cultured pericytes exposed to simulated ischemia for 12 h in vitro contracted after less than 1 h, which was about 7 h prior to cell death. Rapamycin significantly reduced the rate of pericyte contraction during ischemia; however, it did not have a significant effect on pericyte viability at any time point. Rapamycin appeared to reduce pericyte contraction through a mechanism that is independent of changes in intracellular calcium. Using a mouse model of middle cerebral artery occlusion, we showed that rapamycin significantly increased the diameter of capillaries underneath pericytes and increased the number of open capillaries 30 min following recanalisation. Our findings suggest that rapamycin may be a useful adjuvant therapeutic to reduce pericyte contraction and improve cerebral reperfusion post-stroke.

Continued dysfunction of capillary pericytes promotes no-reflow after experimental stroke in vivo.

Incomplete reperfusion of the microvasculature ('no-reflow') after ischaemic stroke damages salvageable brain tissue. Previous ex vivo studies suggest pericytes are vulnerable to ischaemia and may exacerbate no-reflow, but the viability of pericytes and their association with no-reflow remains under-explored in vivo. Using longitudinal in vivo two-photon single-cell imaging over 7 days, we showed that 87% of pericytes constrict during cerebral ischaemia and remain constricted post reperfusion, and 50% of the pericyte population are acutely damaged. Moreover, we revealed ischaemic pericytes to be fundamentally implicated in capillary no-reflow by limiting and arresting blood flow within the first 24 h post stroke. Despite sustaining acute membrane damage, we observed that over half of all cortical pericytes survived ischaemia and responded to vasoactive stimuli, upregulated unique transcriptomic profiles and replicated. Finally, we demonstrated the delayed recovery of capillary diameter by ischaemic pericytes after reperfusion predicted vessel reconstriction in the subacute phase of stroke. Cumulatively, these findings demonstrate that surviving cortical pericytes remain both viable and promising therapeutic targets to counteract no-reflow after ischaemic stroke.

Inhibition of Fli‐1 attenuates inflammation, pericyte loss and cognitive deficits in Alzheimer’s disease

AbstractBackgroundBrain pericytes maintain the integrity of the blood‐brain barrier (BBB) and facilitate the removal of amyloid β (Aβ) which is critical to healthy brain activity (Ethan et al., Brain Pathol., 24: 371‐386, 2014). Pericyte degeneration has been observed in brains from patients with Alzheimer’s disease (AD) and animal models (Jesse et al., Brain Pathol., 23: 303‐10, 2013; Wu et al., Aging (Albany NY), 11: 6120‐6133, 2019). Our previous data demonstrated that friend leukemia virus integration 1 (Fli‐1), an ETS transcription factor, regulates pericyte viability and vascular injury in murine sepsis (Li et al., J Infect Dis, 218: 1995‐2005, 2018); however, the role of Fli‐1 in AD remains unknown.MethodsFli‐1 levels and pericyte number were detected in postmortem brains from 21 patients with AD and 17 healthy controls, and in 5xFAD transgenic mice. Control or Fli‐1 antisense oligonucleotide Gapmers were injected into the hippocampus of 5xFAD mice at 3 and 4.5 months of age. Pericyte number, inflammatory mediators, Aβ deposition, BBB function and cognitive deficits were evaluated at 6 months of age. Human brain pericytes were transfected with control or Fli‐1 antisense Gapmers for 48 h and cultured with or without freshly aggregated Aβ40 for 7 consecutive days. Cell viability and apoptosis were detected.ResultsWe demonstrated that Fli‐1 expression was increased in postmortem brains from a cohort of human AD donors and in 5xFAD mice, which corresponded with a decreased pericyte number, elevated inflammatory mediators, and increased Aβ accumulation as compared to cognitively normal individuals and WT mice, respectively. Antisense oligonucleotide Fli‐1 Gapmer administrated via intrahippocampal injection decelerated pericyte loss, reduced inflammatory responses, ameliorated cognitive deficits, improved BBB dysfunction, and decreased Aβ deposition in 5xFAD mice. Fli‐1 Gapmer‐mediated inhibition of Fli‐1 protected against Aβ accumulation induced human brain pericyte apoptosis in vitro.ConclusionThese composite results indicate that Fli‐1 contributes to pericyte loss, inflammatory response, Aβ deposition, vascular dysfunction and cognitive decline, and suggest that suppression of Fli‐1 may represent a novel therapeutic strategy for AD.

Association of Placental Growth Factor and Angiopoietin in Human Retinal Endothelial Cell-Pericyte co-Cultures and iPSC-Derived Vascular Organoids

PurposePlacental growth factor (PlGF) and Angiopoietin (Ang)-1 are two proteins that are involved in the regulation of endothelial cell (EC) growth and vasculature formation. In the retina and endothelial cells, pericytes are the major source of both molecules. The purpose of this study is to examine the association of PlGF and Ang-1 with human EC/pericyte co-cultures and iPSC-derived vascular organoids.MethodsIn this study, we used co-cultures of human primary retinal endothelial cells (HREC) and primary human retinal pericytes (HRP), western blotting, immunofluorescent analysis, TUNEL staining, LDH-assays, and RNA seq analysis, as well as human-induced pluripotent stem cells (iPSC), derived organoids (VO) to study the association between PlGF and Ang-1.ResultsInhibition of PlGF by PlGF neutralizing antibody in HREC-HRP co-cultures resulted in the increased expression of Ang-1 and Tie-2 in a dose-dependent manner. This upregulation was not observed in HREC and HRP monocultures but only in co-cultures suggesting the association of pericytes and endothelial cells. Furthermore, Vascular endothelial growth factor receptor 1 (VEGFR1) inhibition abolished the Ang-1 and Tie-2 upregulation by PlGF inhibition. The pericyte viability in high-glucose conditions was also reduced by VEGFR1 neutralization. Immunofluorescent analysis showed that Ang-1 and Ang-2 were expressed mainly by perivascular cells in the VO. RNA seq analysis of the RNA isolated from VO in high glucose conditions indicated increased PlGF and Ang-2 expressions in the VO. PlGF inhibition increased the expression of Ang-1 and Tie-2 in VO, increasing the pericyte coverage of the VO microvascular network.ConclusionCombined, these results suggest PlGF's role in the regulation of Ang-1 and Tie-2 expression through VEGFR1. These findings provide new insights into the neovascularization process in diabetic retinopathy and new targets for potential therapeutic intervention.

Components of Salvia miltiorrhiza and Panax notoginseng Protect Pericytes Against OGD/R-Induced Injury via Regulating the PI3K/AKT/mTOR and JNK/ERK/P38 Signaling Pathways.

Salvia miltiorrhiza (SAL) and Panax notoginseng (PNS) are widely used in treating of ischemic stroke. However, it is unknown which components of SAL and PNS protect brain microvascular pericytes after an ischemic stroke. We evaluated the protective effects and mechanisms of SAL and PNS components in pericytes subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Pericytes were subjected to OGD/R. Cell Counting Kit-8 (CCK-8) was usedto evaluate cell viability. ROS and SOD kits were used to detect oxidative stress. Flow cytometry was performed to analyze cell apoptosis. To evaluate cell migration, a scratch assay was performed. Expression of cleaved caspase-3, Bcl-2, Bax, VEGF, Ang-1, PDGFR-β, PI3K/AKT/mTOR, and JNK/ERK/P38 signaling pathways were identified using western blot. The results revealed that salvianolic acid B (Sal B), salvianolic acid D (Sal D), notoginsenoside R1 (R1), ginsenoside Rb1 (Rb1), and ginsenoside Rg1 (Rg1) increased the cell viability of pericytes subjected to OGD/R, reduced the level of ROS, and increased the expression of SOD. The components reduced cell apoptosis, increased the protein level of Bcl-2/Bax, reduced the level of cleaved caspase-3/caspase-3, increased cell migration, and enhanced the levels of Ang-1, PDGFR-β, and VEGF. The components could activate PI3K/AKT/mTOR pathway while inhibiting the JNK/ERK/P38 pathway. Studies found that Sal B, Sal D, R1, Rb1, and Rg1 inhibited oxidative stress and apoptosis while increasing the release of pro-angiogenic regulators of pericytes related to the PI3K/AKT/mTOR and JNK/ERK/P38 signaling pathways. This provides a potential foundation for developing monomeric drugs for treating ischemic stroke.

Suppression of Fli-1 protects against pericyte loss and cognitive deficits in Alzheimer's disease

Brain pericytes regulate cerebral blood flow, maintain the integrity of the blood-brain barrier (BBB), and facilitate the removal of amyloid β (Aβ), which is critical to healthy brain activity. Pericyte loss has been observed in brains from patients with Alzheimer's disease (AD) and animal models. Our previous data demonstrated that friend leukemia virus integration 1 (Fli-1), an erythroblast transformation-specific (ETS) transcription factor, governs pericyte viability in murine sepsis; however, the role of Fli-1 and its impact on pericyte loss in AD remain unknown. Here, we demonstrated that Fli-1 expression was up-regulated in postmortem brains from a cohort of human AD donors and in 5xFAD mice, which corresponded with a decreased pericyte number, elevated inflammatory mediators, and increased Aβ accumulation compared with cognitively normal individuals and wild-type (WT) mice. Antisense oligonucleotide Fli-1 Gapmer administered via intrahippocampal injection decelerated pericyte loss, decreased inflammatory response, ameliorated cognitive deficits, improved BBB dysfunction, and reduced Aβ deposition in 5xFAD mice. Fli-1 Gapmer-mediated inhibition of Fli-1 protected against Aβaccumulation-induced human brain pericyte apoptosis invitro. Overall, these studies indicate that Fli-1 contributes to pericyte loss, inflammatory response, Aβ deposition, vascular dysfunction, and cognitive decline, and suggest that inhibition of Fli-1 may represent novel therapeutic strategies for AD.

MiR-485-5p alleviates Alzheimer's disease progression by targeting PACS1.

Alzheimer’s disease (AD) is a common dementia and a heterogeneous disease. Previous research has validated that microRNAs (miRNAs) are pivotal regulators in the initiation and development of tremendous diseases including AD. MicroRNA-485-5p (miR-485-5p) was reported to be an important participant implicated in several neurological diseases, but its role in AD still needs to be further investigated. In this research, we explored the biological function of miR-485-5p in AD. RT-qPCR revealed that miR-485-5p expression was downregulated in the hippocampus of APP/PS1 mice. Additionally, miR-485-5p overexpression facilitated the learning and memory capabilities of APP/PS1 mice according to Morris water maze test, fear conditioning test, and immunofluorescent staining. Moreover, CCK-8 assay, flow cytometric analysis, and western blot analysis suggested that miR-485-5p overexpression promoted pericyte viability and prohibited pericyte apoptosis in APP/PS1 mice. Mechanistically, miR-485-5p directly targeted PACS1 in pericytes, as shown in a luciferase reporter assay. In rescue assays, PACS1 overexpression countervailed the effect of miR-485-5p overexpression on pericyte viability and apoptosis. In conclusion, miR-485-5p ameliorates AD progression by targeting PACS1.

Lenvatinib Targets PDGFR-β Pericytes and Inhibits Synergy With Thyroid Carcinoma Cells: Novel Translational Insights.

Pericyte populations abundantly express tyrosine kinases (eg, platelet-derived growth factor receptor-β [PDGFR-β]) and impact therapeutic response. Lenvatinib is a clinically available tyrosine kinase inhibitor that also targets PDGFR-β. Duration of therapeutic response was shorter in patients with greater disease burden and metastasis. Patients may develop drug resistance and tumor progression. Develop a gene signature of pericyte abundance to assess with tumor aggressiveness and determine both the response of thyroid-derived pericytes to lenvatinib and their synergies with thyroid carcinoma-derived cells. Using a new gene signature, we estimated the relative abundance of pericytes in papillary thyroid carcinoma (PTC) and normal thyroid (NT) TCGA samples. We also cocultured CD90+;PAX8- thyroid-derived pericytes and BRAFWT/V600E-PTC-derived cells to determine effects of coculture on paracrine communications and lenvatinib response. Pericyte abundance is significantly higher in BRAFV600E-PTC with hTERT mutations and copy number alterations compared with NT or BRAFWT-PTC samples, even when data are corrected for clinical-pathologic confounders. We have identified upregulated pathways important for tumor survival, immunomodulation, RNA transcription, cell-cycle regulation, and cholesterol metabolism. Pericyte growth is significantly increased by platelet-derived growth factor-BB, which activates phospho(p)-PDGFR-β, pERK1/2, and pAKT. Lenvatinib strongly inhibits pericyte viability by down-regulating MAPK, pAKT, and p-p70S6-kinase downstream PDGFR-β. Critically, lenvatinib significantly induces higher BRAFWT/V600E-PTC cell death when cocultured with pericytes, as a result of pericyte targeting via PDGFR-β. This is the first thyroid-specific model of lenvatinib therapeutic efficacy against pericyte viability, which disadvantages BRAFWT/V600E-PTC growth. Assessing pericyte abundance in patients with PTC could be essential to selection rationales for appropriate targeted therapy with lenvatinib.

An improved method of culturing somatotropic cells from rat adenohypophysis

This study aimed to propose a simple and practical method for culturing primary rat somatotropic cells in vitro free of pericytes contamination. Rat adenohypophyses were randomly divided into two groups. An improved method was used in group A (digesting adenohypophysis with 0.25% trypsin-EDTA, followed by removing pericytes by double filtration and using serum-free medium for culturing somatotropic cells). The traditional method was used in group B (digesting adenohypophysis with 0.35% collagenase, using serum medium for culturing somatotropic cells, and removing pericytes by changing the culture dish). The numbers and viability of somatotropic cells were higher in group A than in group B after 6 days. GH secretion of somatotropic cells was also higher in group A than in group B. Besides, the pericytes grew rapidly only in group B after 3 days. α-SMA, type I collagen, and type III collagen had weaker expression in group A. Also, the viability of pericytes decreased in group A. The improved method could solve the problem of pericytes contamination, and the culture of primary rat somatotropic cells in vitro was successful. This method can be used for other primary cultures with pericytes contamination.

Amyloid‐beta 1‐40 is associated with alterations in NG2+ pericyte population ex vivo and in vitro

SummaryThe population of brain pericytes, a cell type important for vessel stability and blood brain barrier function, has recently been shown altered in patients with Alzheimer's disease (AD). The underlying reason for this alteration is not fully understood, but progressive accumulation of the AD characteristic peptide amyloid‐beta (Aβ) has been suggested as a potential culprit. In the current study, we show reduced number of hippocampal NG2+ pericytes and an association between NG2+ pericyte numbers and Aβ1‐40 levels in AD patients. We further demonstrate, using in vitro studies, an aggregation‐dependent impact of Aβ1‐40 on human NG2+ pericytes. Fibril‐EP Aβ1‐40 exposure reduced pericyte viability and proliferation and increased caspase 3/7 activity. Monomer Aβ1‐40 had quite the opposite effect: increased pericyte viability and proliferation and reduced caspase 3/7 activity. Oligomer‐EP Aβ1‐40 had no impact on either of the cellular events. Our findings add to the growing number of studies suggesting a significant impact on pericytes in the brains of AD patients and suggest different aggregation forms of Aβ1‐40 as potential key regulators of the brain pericyte population size.

Amylin alters human brain pericyte viability and NG2 expression

Amylin, a pancreatic β-cell-derived peptide hormone, forms inclusions in brain microvessels of patients with dementia who have been diagnosed with type 2 diabetes and Alzheimer’s disease. The cellular localization of these inclusions and the consequences thereof are not yet known. Using immunohistochemical staining of hippocampus and parahippocampal cortex from patients with Alzheimer’s disease and non-demented controls, we show that amylin cell inclusions are found in pericytes. The number of amylin cell inclusions did not differ between patients with Alzheimer’s disease and controls, but amylin-containing pericytes displayed nuclear changes associated with cell death and reduced expression of the pericyte marker neuron-glial antigen 2. The impact of amylin on pericyte viability was further demonstrated in in vitro studies, which showed that pericyte death increased in presence of fibril- and oligomer amylin. Furthermore, oligomer amylin increased caspase 3/7 activity, reduced lysate neuron-glial antigen 2 levels and impaired autophagy. Our findings contribute to increased understanding of how aggregated amylin affects brain vasculature and highlight amylin as a potential factor involved in microvascular pathology in dementia progression.

Abstract 17867: The Pro-angiogenic Long Non-coding Rna Tykril Controls Human Pericyte Function Through Regulation Of P53 Activity and Pdgfrß Expression

Rationale: Pericytes contribute to the formation, maturation and stabilization of blood vessels, making them indispensable for proper cardiovascular organ function. Although long non-coding RNAs (lncRNAs) are pivotal in cardiovascular development, their role in pericytes has so far been neglected. Objective: Here we assessed the hypothesis that lncRNAs significantly regulate pericyte function. Methods and Results: Via RNA deep sequencing we show that the hypoxia induced lncRNA TYKRIL (Tyrosine Kinase Receptor Inducing lncRNA) modulates expression of the tumor suppressor p53 and Platelet derived growth factor receptor beta (PDGFRß) in human pericytes. Luciferase reporter arrays demonstrated that silencing of TYKRIL with GapmeRs (0.22+0.04 vs. scramble Ctrl., n=8, P<0.001) significantly enhances activity of p53 (2.9+0.55 fold induction vs. scramble Ctrl., n=3, P=0.0103). TYKRIL knockdown increased acetylation of the p53 lysine residue K382, which is known to be required for p53 activation. TYKRIL silencing downregulated the known p53 target PDGFRß (protein expression: 0.46+0.08 vs. Ctrl., n=4, P<0.001) which consecutively disrupted AKT and ERK1/2 phosphorylation. Thereby, pericyte viability (MTT cell viability: 0.8+0.03 vs. Ctrl., n=3, P<0.01), proliferation (Ki67 proliferation index fold change 0.71+0.05 vs. Ctrl., n=6, P<0.001) and recruitment towards endothelial cells in matrigel co-culture assays was markedly reduced (fold change 0.37+0.03 vs. Ctrl., n=5-7, P<0.001). In turn, silencing of p53 reversed these effects (MTT cell viability: TYKRIL knockdown + scramble 0.7+0.04; TYKRIL knockdown + siRNAp53 0.94+0.04, n=11-12, P<0.01). In patients with ischemic heart failure (n=18), cardiac TYKRIL and PDGFRß expression are significantly reduced compared to controls (TYKRIL vs. Ctrl. 0.01+0.005, P<0.001; PDGFRß mRNA expression: 0.44+0.11, P<0.05). Conclusion: Our results identify TYKRIL as a pro-angiogenic lncRNA that is down-regulated by ischemic heart failure and significantly regulates pericyte viability and recruitment towards endothelial cells by repression of p53 activity and stabilization of PDGFRß signalling.

Selective eradication of tumor vascular pericytes by peptide-conjugated nanoparticles for antiangiogenic therapy of melanoma lung metastasis

Antiangiogenic cancer therapy based on nanoparticulate drug delivery systems (nano-DDS) is emerging as a promising new approach besides the proved molecular-targeted antiangiogenic agents. The current nano-DDS are restricted to the targeting to tumor vascular endothelial cells, but seldom efforts have been made to target the tumor vascular pericytes which are also actively involved in tumor angiogenesis. In this study, we developed a new nano-DDS, TH10 peptide (TAASGVRSMH) conjugated nanoparticles loading docetaxel (TH10-DTX-NP) that can target the NG2 proteoglycan highly expressed in tumor vascular pericytes, for the investigation of therapeutic efficacy in the mice bearing B16F10-luc-G5 melanoma experimental lung metastasis. The results demonstrated that TH10-DTX-NP achieved controlled drug release in PBS and the mixture of rat plasma and PBS (1:1, v/v), and exhibited favorable in vivo long-circulating feature. TH10 peptide conjugation facilitated the nanoparticle internalization in pericytes via the interaction between TH10 and NG2 receptor, leading to more inhibition of pericyte viability and migration. TH10-conjugated nanoparticles could accurately target the vascular pericytes of B16F10-luc-G5 lung metastasis, where DTX-induced pronounceable pericyte apoptosis. TH10-DTX-NP significantly prolonged the mice survival with no obvious toxicity, and this enhanced antitumor effect was closely related with the decreased pericyte density and microvessel density in the lung metastases. The present research reveals the potency and significance of targeting tumor vascular pericytes using nano-DDS in antiangiogenic cancer therapy.

The Antiangiogenic 16K Prolactin Impairs Functional Tumor Neovascularization by Inhibiting Vessel Maturation

BackgroundAngiogenesis, the formation of new blood vessels from existing vasculature, plays an essential role in tumor growth, invasion, and metastasis. 16K hPRL, the antiangiogenic 16-kDa N-terminal fragment of human prolactin was shown to prevent tumor growth and metastasis by modifying tumor vessel morphology.Methodology/Principal FindingsHere we investigated the effect of 16K hPRL on tumor vessel maturation and on the related signaling pathways. We show that 16K hPRL treatment leads, in a murine B16-F10 tumor model, to a dysfunctional tumor vasculature with reduced pericyte coverage, and disruption of the PDGF-B/PDGFR-B, Ang/Tie2, and Delta/Notch pathways. In an aortic ring assay, 16K hPRL impairs endothelial cell and pericyte outgrowth from the vascular ring. In addition, 16K hPRL prevents pericyte migration to endothelial cells. This event was independent of a direct inhibitory effect of 16K hPRL on pericyte viability, proliferation, or migration. In endothelial cell-pericyte cocultures, we found 16K hPRL to disturb Notch signaling.Conclusions/SignificanceTaken together, our data show that 16K hPRL impairs functional tumor neovascularization by inhibiting vessel maturation and for the first time that an endogenous antiangiogenic agent disturbs Notch signaling. These findings provide new insights into the mechanisms of 16K hPRL action and highlight its potential for use in anticancer therapy.

Regulation of extracellular-superoxide dismutase in rat retina pericytes

Diabetic retinopathy (DR) is regarded as a disease of the retinal microvascular system and metabolic abnormalities that are characteristic of oxidative stress and endoplasmic reticulum (ER) stress have been identified in the retina. Pericytes are known to be susceptible to oxidative stress and selective dropout of pericytes is one of the earliest pathological changes in DR. Extracellular-superoxide dismutase (EC-SOD) is a major antioxidative enzyme and protects vascular cells from the damaging effects of superoxide. Treatment with own conditioned medium significantly decreased EC-SOD expression in pericytes, while the expression of vascular endothelial growth factor and tumor necrosis factor-α were elevated. The addition of chemical chaperone 4-phenyl butyric acid significantly suppressed the effects of conditioned medium on EC-SOD and GRP78, a prominent ER-resident chaperone. Moreover, the cell viability of pericytes changed in a manner similar to that of EC-SOD expression. These results suggest that the expressions of EC-SOD should be regulated, at least partially, through ER stress. Continuous flow of culture media neutralized the ER-stress triggered decrease of EC-SOD expression. The stagnation of factors related to ER-stress around pericytes might reduce EC-SOD expression under pathophysiological conditions such as retinal edema, and this could induce and/or promote the intraretinal microvascular impairment and development of pathogenesis in DR.

RATIONALE FOR COMBINATION THERAPY IN AGE-RELATED MACULAR DEGENERATION

Choroidal neovascularization (CNV) is typically characterized as an invasion of blood vessels, but it is important to recognize concurrent inflammatory and mesenchymal cell infiltration. A two component model of CNV can be thought to be composed of a vascular component and an extravascular component. Macular damage is possible through either. Given the redundancy and complex interaction among biologic systems involved in the production of CNV, it is likely that a monotherapeutic approach will not be fully effective. The vascular component of CNV arises through the orchestrated interaction of a number of growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), which maintains pericyte viability. The vascular component of CNV can be selectively targeted with anti-VEGF and anti-PDGF drugs or nonselectively with such modalities as ionizing radiation. The extravascular component can be targeted selectively by inhibiting specific cytokines such as tumor necrosis factor alpha or nonselectively with antiinflammatory drugs such as corticosteroids.

Effect of pigment epithelium derived factor on microvascular cells

Objectives: Angiogenesis is a key process of tumor growth and metastasis. Recently, pigment epithelium derived factor (PEDF) is known as a potent antiangiogenic factor. We examined the expression of PEDF in human endothelial cells and pericytes as microvascular cells. Further we examined the effects of exogenous PEDF on endothelial cell and pericyte proliferation. Methods: Pericytes utilized in this study were human retinal capillary pericytes. Endothelial cells were human umbilical vein endothelial cells (HUVEC). The mRNA expression of PDGF and c-fos was examined by RT-PCR. Signal transduction assays were done with Western blot. Cell proliferation was evaluated by DNA synthesis (BrdU assay). Results: Pericytes expressed the high levels of PDGF mRNA while the expression of PEDF in endothelial cells was almost undetectable. PEDF stimulates pericyte proliferation at both low (0.1–1.0 μg/mL) and high (50 μg/mL) doses. The stimulation by PEDF also activated MAPK and CREB and induced early response gene c-fos mRNA expression. In contrast, the effects of exogenous PEDF on endothelial cells were dose-dependent. PEDF increased cell proliferation at the low doses (0.1–1.0 μg/ml) while cell proliferation was significantly decreased with high (50 μg/ml) dose of PEDF. More than 80% of cells treated with the high dose of PEDF were TUNEL positive, suggesting that endothelial cells undergo apoptosis under the high concentration of PEDF. Conclusion: Pericyte is an important source of PEDF. In physiological condition, PEDF enhances pericyte viability and prevent endothelial cell proliferation. In the environment with low levels of PEDF, endothelial cell proliferation can be easily induced.

Inhibition of Platelet-Derived Growth Factor Promotes Pericyte Loss and Angiogenesis in Ischemic Retinopathy

We investigated whether inhibition of platelet-derived growth factor (PDGF) receptor tyrosine kinase activity would affect pericyte viability, vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR-2) expression and angiogenesis in a model of retinopathy of prematurity (ROP). ROP was induced in Sprague Dawley rats by exposure to 80% oxygen from postnatal (P) days 0 to 11 (with 3 hours/day in room air), and then room air from P12-18 (angiogenesis period). Shams were neonatal rats in room air from P0-18. STI571, a potent inhibitor of PDGF receptor tyrosine kinase, was administered from P12-18 at 50 or 100 mg/kg/day intraperitoneal (i.p.). Electron microscopy revealed that pericytes in the inner retina of both sham and ROP rats appeared normal; however STI571 induced a selective pericyte and vascular smooth muscle degeneration. Immunolabeling for caspase-3 and alpha-smooth muscle cell actin in consecutive paraffin sections of retinas confirmed that these degenerating cells were apoptotic pericytes. In all groups, VEGF and VEGFR-2 gene expression was located in ganglion cells, the inner nuclear layer, and retinal pigment epithelium. ROP was associated with an increase in both VEGF and VEGFR-2 gene expression and blood vessel profiles in the inner retina compared to sham rats. STI571 at both doses increased VEGF and VEGFR-2 mRNA and exacerbated angiogenesis in ROP rats, and in sham rats at 100 mg/kg/day. In conclusion, PDGF is required for pericyte viability and the subsequent prevention of VEGF/VEGFR-2 overexpression and angiogenesis in ROP.

A study of capillary pericyte viability on extracellular matrix produced by endothelial cells in high glucose.

Thickening of the basement membrane and selective loss of pericytes occur early in diabetic retinopathy. As we showed previously that pericyte adhesion is impaired on extracellular matrix produced by endothelial cells in high hexose concentrations, we aimed to verify if altered adhesion could influence pericyte viability and replication. Conditioned extracellular matrices were obtained by growing human umbilical vein endothelial cells in media containing 28 mmol/l D-glucose, with or without the inhibitors of protein glycation thiamine or aminoguanidine, and D-galactose or L-glucose up to 28 mmol/l. Having removed the endothelium, bovine retinal pericytes were grown on these matrices and, in separate experiments, on laminin, fibronectin or type IV collagen. Pericyte viability and replication were measured by cell counts and DNA synthesis after 7 days, cell cycle traversal after 2 days and apoptosis after 18 h, 2 days and 7 days. Pericyte counts and DNA synthesis were reduced on matrices produced in high D-glucose and D-galactose, whilst matrix obtained in L-glucose reduced DNA synthesis but not counts. Both thiamine and aminoguanidine corrected reduced pericyte viability when added to high D-glucose. Cell cycle and apoptosis were not affected by growing pericytes on different conditioned matrices. Laminin, fibronectin and type IV collagen did not modify pericyte replication. Reduced pericyte counts could depend on impaired initial adhesion to the extracellular matrix produced by endothelium in high hexose concentrations, rather than impaired replication or viability. Altered cell-matrix interactions might facilitate pericyte dropout in diabetic retinopathy, independently of the effects of high glucose on pericyte replication.