Pericyte Apoptosis Research Articles

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.

Retinal vascular abnormalities and blood-retinal barrier breakdown in Alzheimer's disease.

Vascular changes including cerebral amyloid angiopathy (CAA), reduced blood flow, pericyte loss and vascular platelet-derived growth factor receptor-β (PDGFRβ) deficiency have emerged as prominent features of Alzheimer's disease (AD). We previously identified the existence of the pathological hallmarks of AD, amyloid β-protein (Aβ) plaques, along with intracellular and vascular Aβ deposits in the neurosensory retina of AD patients. Data suggest that certain retinal signs manifest early in disease progression and appear similar to those observed in the brain. Yet, retinal vascular abnormalities in AD and their ability to predict cerebral pathology and cognitive decline are poorly understood. Here, we assessed vascular changes including capillary degeneration, PDGFRβ expression, vascular amyloidosis, pericyte loss, and blood-retinal barrier (BRB) permeability in postmortem retinas from a cohort of mild cognitively impaired (MCI) and AD patients. We evaluated retinal cross-sections from pre-defined topographical regions and isolated retinal vascular networks labeled by periodic acid-Schiff or immunofluorescence. We further quantified capillary degeneration, PDGFRβ expression, vascular amyloidosis, and inner BRB tight-junction molecule expression and BRB leakage in transgenic APPSWE /PS1ΔE9 mouse models of AD (ADtg mice) at different disease stages. Early and progressive PDGFRβ deficiency and pericyte loss were found in retinas from AD patients. PDGFRβ deficiency associated with vascular Aβ deposits in postmortem retinas from a cohort of MCI and AD patients. In ADtg mice as compared to age- and sex-matched wild-type mice, we found significant degeneration of retinal capillaries (P<0.0001). ADtg retinas also displayed a substantial vascular PDGFRβ deficiency (∼50% reduction, P=0.0017) and vascular Aβ deposition, which inversely correlated with the extent of degenerated capillaries (Pearson's r=-0.8, P=0.0016). Tight-junction alterations including claudin-1 downregulation and increased BRB permeability were found in ADtg mice. In postmortem retinas from the human cohort, retinal PDGFRβ loss significantly associated with increased retinal vascular Aβ40 and Aβ42 burdens. Decreased vascular LRP-1 and early apoptosis of pericytes in AD retina were further revealed. Correlations were identified between retinal vascular abnormalities and cerebral Aβ burden, CAA, and clinical status. Overall, the identification of age- and Alzheimer's-dependent retinal capillary degeneration and compromised BRB integrity starting at early disease stages provide novel targets for AD diagnosis and therapy.

Pathomorphological changes of capillaries in the cerebral cortex in type 2 diabetes mellitus

The aim of the study was to determine microscopic, immunohistochemical, electron microscopic, morphometric parameters of capillaries in the cerebral cortex in deceased patients with type 2 diabetes mellitus (DM) in comparison with the conditional control group and the group of deceased patients with dyscirculatory ischemic encephalopathy. Materials and methods. Microscopic, immunohistochemical, electron microscopic and morphometric studies of the cerebral cortex capillaries were performed in 3 groups: group I – 20 deceased patients with type 2 DM, group II conditional control (CC) – 20 deceased patients without clinical morphological signs of DM and cerebrovascular pathology, group III – 20 patients with dyscirculatory ischemic encephalopathy (DIEP). Results. It was found that in type 2 DM, the capillaries in the cerebral cortex lose pericytes due to their apoptosis: the number of pericytes in the cortical capillaries in type 2 DM was statistically significantly lower by 57.14 % compared to CC group and 50.00 % lower compared to DIEP group, the level of caspase-3 expression in the cortical microvessels in type 2 DM was significantly higher by 230.85 % compared with CC group and 81.67 % higher than in DIEP group. According to electron microscopy in type 2 DM, apoptosis of pericytes and single endothelial cells in the cerebral cortex capillaries was determined as well as significant expansion of basement membranes with the accumulation of electron-dense amorphous material and collagen fibrils. According to the results of morphometry, the outer diameter of the cortical capillaries in type 2 DM group was 4.90 % significantly larger, the inner diameter was 9.78 % smaller and the walls were 66.62 % thicker (compared with CC group) due to the accumulation of PAS-positive substances of blood serum and fibrosis, confirmed by 22.96 % greater area of type IV collagen expression in the microvessel walls. Conclusions. The pathomorphological changes of microvessels identified in deceased patients with type 2 diabetes mellitus are signs of diabetic cerebral microangiopathy.

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.

Prostaglandin F2α protects against pericyte apoptosis by inhibiting the PI3K/Akt/GSK3β/β-catenin signaling pathway.

BackgroundDiabetic retinopathy (DR) is the most common microvascular complication of diabetes and the main cause of non-traumatic blindness in adults. Pericyte loss is known to be an early pathological change of DR. Our group’s previous research indicated that prostaglandin F2α (PGF2α) acts as an eicosanoidal protector against non-proliferative DR that can regulate the mobility of pericytes in a RhoA-mediated manner. However, the effect of PGF2α on pericyte apoptosis has yet to be described.MethodsTwo animal models were constructed: a high-fat diet (HFD) and streptozotocin (STZ)-induced type 2 diabetes mouse model and a spontaneous type 2 diabetes db/db mouse model. We analyzed pathological changes, and performed TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining and western blot to detect apoptosis in the retinas of diabetic mice. For our in vitro experiments, we selected human retinal pericytes and subjected them to high-glucose (HG), PGF2α, and AL8810 (an antagonist of the PGF2α receptor) treatment. Subsequently, apoptosis and the levels of PI3K/Akt/GSK3β/β-catenin pathway-related proteins were detected by TUNEL staining and western blot, respectively.ResultsThe levels of apoptosis were increased in the retinas of diabetic mice in both T2DM models. In vitro, HG treatment increased apoptosis and inhibited PI3K/Akt/GSK3β/β-catenin signaling in pericytes. In contrast, PGF2α treatment inhibited pericyte apoptosis while increasing the levels of the PI3K, p-Akt/t-Akt, p-GSK3β/t-GSK3β, and β-catenin proteins; however, these PGF2α-induced effects were eliminated by ALL80.ConclusionsPGF2α may make a key contribution to reducing pericyte apoptosis and protecting against DR via its inhibition of the PI3K/Akt/GSK3β/β-catenin signaling pathway.

RGS5-TGFβ-Smad2/3 axis switches pro- to anti-apoptotic signaling in tumor-residing pericytes, assisting tumor growth.

Regulator-of-G-protein-signaling-5 (RGS5), a pro-apoptotic/anti-proliferative protein, is a signature molecule of tumor-associated pericytes, highly expressed in several cancers, and is associated with tumor growth and poor prognosis. Surprisingly, despite the negative influence of intrinsic RGS5 expression on pericyte survival, RGS5highpericytes accumulate in progressively growing tumors. However, responsible factor(s) and altered-pathway(s) are yet to report. RGS5 binds with Gαi/q and promotes pericyte apoptosis in vitro, subsequently blocking GPCR-downstream PI3K-AKT signaling leading to Bcl2 downregulation and promotion of PUMA-p53-Bax-mediated mitochondrial damage. However, within tumor microenvironment (TME), TGFβ appeared to limit the cytocidal action of RGS5 in tumor-residing RGS5highpericytes. We observed that in the presence of high RGS5 concentrations, TGFβ-TGFβR interactions in the tumor-associated pericytes lead to the promotion of pSmad2-RGS5 binding and nuclear trafficking of RGS5, which coordinately suppressed RGS5-Gαi/q and pSmad2/3-Smad4 pairing. The RGS5-TGFβ-pSmad2 axis thus mitigates both RGS5- and TGFβ-dependent cellular apoptosis, resulting in sustained pericyte survival/expansion within the TME by rescuing PI3K-AKT signaling and preventing mitochondrial damage and caspase activation. This study reports a novel mechanism by which TGFβ fortifies and promotes survival of tumor pericytes by switching pro- to anti-apoptotic RGS5 signaling in TME. Understanding this altered RGS5 signaling might prove beneficial in designing future cancer therapy.

Effect of Shuangdan Mingmu capsule, a Chinese herbal formula, on oxidative stress-induced apoptosis of pericytes through PARP/GAPDH pathway

BackgroundDiabetic retinopathy (DR) has become a worldwide concern because of the rising prevalence rate of diabetes mellitus (DM). Despite much energy has been committed to DR research, it remains a difficulty for diabetic patients all over the world. Since apoptosis of retinal microvascular pericytes (RMPs) is the early characteristic of DR, this study aimed to reveal the mechanism of Shuangdan Mingmu (SDMM) capsule, a Chinese patent medicine, on oxidative stress-induced apoptosis of pericytes implicated with poly (ADP-ribose) polymerase (PARP) / glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pathway.MethodsNetwork pharmacology approach was performed to predict biofunction of components of SDMM capsule dissolved in plasma on DR. Both PARP1 and GAPDH were found involved in the hub network of protein-protein interaction (PPI) of potential targets and were found to take part in many bioprocesses, including responding to the regulation of reactive oxygen species (ROS) metabolic process, apoptotic signaling pathway, and response to oxygen levels through enrichment analysis. Therefore, in vitro research was carried out to validate the prediction. Human RMPs cultured with media containing 0.5 mM hydrogen oxide (H2O2) for 4 h was performed as an oxidative-damage model. Different concentrations of SDMM capsule, PARP1 inhibitor, PARP1 activation, and GAPDH inhibitor were used to intervene the oxidative-damage model with N-Acetyl-L-cysteine (NAC) as a contrast. Flow cytometry was performed to determine the apoptosis rate of cells and the expression of ROS. Cell counting kit 8 (CCK8) was used to determine the activity of pericytes. Moreover, nitric oxide (NO) concentration of cells supernatant and expression of endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), B cell lymphoma 2 (BCL2), vascular endothelial growth factor (VEGF), endothelin 1 (ET1), PARP1, and GAPDH were tested through RT-qPCR, western blot (WB), or immunocytochemistry (ICC).ResultsOverproduction of ROS, high apoptotic rate, and attenuated activity of pericytes were observed after cells were incubated with media containing 0.5 mM H2O2. Moreover, downregulation of SOD, NO, BCL2, and GAPDH, and upregulation of VEGFA, ET1, and PARP1 were discovered after cells were exposed to 0.5 mM H2O2 in this study, which could be improved by PARP1 inhibitor and SDMM capsule in a dose-dependent way, whereas worsened by PARP1 activation and GAPDH inhibitor.ConclusionsSDMM capsule may attenuate oxidative stress-induced apoptosis of pericytes through downregulating PARP expression and upregulating GAPDH expression.

Ghrelin attenuate cerebral microvascular leakage by regulating inflammation and apoptosis potentially via a p38 MAPK-JNK dependent pathway

Ghrelin is a peptide hormone with strong anti-inflammatory properties. In fact, Ghrelin was reported to improve endothelial dysfunction caused by excessive fat. However, its role in preserving the integrity of brain microvascular, under conditions of lipid dysregulation and inflammation, is not known. The objective of this study is to characterize the role of Ghrelin in the protection of cerebral microvascular integrity, during atherosclerosis, and uncover its underlying molecular mechanism. Our results demonstrated that an atherosclerotic condition, brought on by a high fat diet (HFD), can produce massive increases in serum inflammatory factors, blood lipids, cerebral microvascular leakage, and activation of the p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) (p38 MAPK-JNK) pathway. It also produced significantly damaged pericytes morphology, resulting in pericyte decrease. Ghrelin treatment, on the other hand, protected against cerebral microvascular leakage and pericytes damage. Ghrelin effectively downregulated the expression of pro-inflammatory cytokines, and it also suppressed the p38 MAPK-JNK signaling pathway. Additionally, in isolated mouse cerebral microvascular pericytes, ox-LDL lead to increased apoptosis and secretion of inflammatory factors, along with an elevation in phosphorylated p38 MAPK-JNK proteins. Alternately, Ghrelin administration markedly lowered expression of inflammatory factors, suppressed the p38 MAPK-JNK signaling path, and halted cell apoptosis. However, pretreatment of Hesperetin, a p38 MAPK-JNK agonist, abrogated the Ghrelin-mediated suppression of inflammation and apoptosis in pericytes. Taken together, these results suggest that Ghrelin restored cerebral microvascular integrity and reduced vascular leakage in atherosclerosis mice, in part, by its regulation of inflammatory and apoptotic signaling pathways in pericytes.

Interleukin-1β induces pericyte apoptosis via the NF-κB pathway in diabetic retinopathy

Pericytes play a crucial role in preventing endothelial permeability by maintaining the integrity of tight junctions in endothelial cells; however, early pathological change in diabetic retinopathyis pericyte loss, which can lead to visual impairment by increasing endothelial permeability. Therefore, finding proteins and mechanisms that cause pericyte loss in diabetic retinopathy is beneficial for attenuating vision impairment. The present study focused on the effect of IL-1β on pericyte loss and endothelial permeability in diabetic retinopathy. It was demonstrated that IL-1β increased in the diabetic mouse retina and that the source of IL-1β could be endothelial cells and microglia. IL-1β induced pericyte apoptosis via NF-κB activation under high glucose conditions, but did not induce endothelial cell apoptosis. Moreover, IL-1β did not affect permeability in the endothelial cell monolayer; however, when cocultured with pericytes and endothelial cells, it increased endothelial cell permeability by reducing the amount of tight junction protein in endothelial cells. Furthermore, NF-κB inhibitor restored the altered permeability and tight junction protein expression in endothelial cells induced by IL-1β in cocultures of pericytes and endothelial cells. Collectively, IL-1β induced pericyte apoptosis via NF-κB activation under high glucose conditions, thereby increasing endothelial permeability in diabetic retinopathy. Blocking IL-1β/NF-κB signaling could be a promising therapeutic target to prevent pericyte loss in diabetic retinopathy.

Hepatocyte growth factor prevents pericyte loss in diabetic retinopathy

Diabetic retinopathy (DR) is a disease that causes blindness due to vascular leakage or abnormal angiogenesis. Hepatocyte growth factor (HGF) is increased in the serum or vitreous fluid in proliferative diabetic retinopathy (PDR) patients, although the effect of HGF on the blood vessels remains unclear. This study focused on the effect of HGF on pericyte (PC) survival and endothelial cell (EC) permeability. It was demonstrated that HGF was increased in the diabetic mouse retina. However, HGF prevented PC apoptosis caused by TNF-α, which increased in the diabetic retinas both in vitro and in vivo. In addition, HGF was involved in PC survival by increasing the Akt signaling pathway. Moreover, HGF strengthened the EC tight junction in co-cultures of PCs and ECs by promoting PC survival, thereby reducing EC permeability. These results suggest that HGF may play a role in the prevention of increased vascular leakage by inhibiting the PC loss that occurs in DR to some extent. However, careful HGF reduction in DR might avoid an increase in PC loss.

Deficiency of Notch signaling in pericytes results in arteriovenous malformations.

Arteriovenous malformations (AVMs) are high-flow lesions directly connecting arteries and veins. In the brain, AVM rupture can cause seizures, stroke, and death. Patients with AVMs exhibit reduced coverage of the vessels by pericytes, the mural cells of microvascular capillaries; however, the mechanism underlying this pericyte reduction and its association with AVM pathogenesis remains unknown. Notch signaling has been proposed to regulate critical pericyte functions. We hypothesized that Notch signaling in pericytes is crucial to maintain pericyte homeostasis and prevent AVM formation. We inhibited Notch signaling specifically in perivascular cells and analyzed the vasculature of these mice. The retinal vessels of mice with deficient perivascular Notch signaling developed severe AVMs, together with a significant reduction in pericytes and vascular smooth muscle cells (vSMC) in the arteries, while vSMCs were increased in the veins. Vascular malformations and pericyte loss were also observed in the forebrain of embryonic mice deficient for perivascular Notch signaling. Moreover, the loss of Notch signaling in pericytes downregulated Pdgfrb levels and increased pericyte apoptosis, pointing to a critical role for Notch in pericyte survival. Overall, our findings reveal a mechanism of AVM formation and highlight the Notch signaling pathway as an essential mediator in this process.

Microtubule Stabilization Promotes Microcirculation Reconstruction After Spinal Cord Injury

Spinal cord microcirculation plays an important role in maintaining the function of spinal cord neurons and other cells. Previous studies have largely focused on the ability of microtubule stabilization to inhibit the fibroblast migration and promote axon regeneration after spinal cord injury (SCI). However, the effect of microtubule stabilization treatment on microcirculation reconstruction after SCI remains unclear. By using immunofluorescence, we found that microtubule stabilization treatment improved microcirculation reconstruction via increasing the number of microvessels, pericytes, and the perfused microvessels after SCI. To clarify the underlying mechanisms, rat brain microvascular endothelial cells and pericytes were subjected to glucose oxygen deprivation. By using flow cytometry and western blotting, we found that microtubule stabilization treatment inhibited apoptosis and migration of endothelial cells and pericytes but promoted proliferation and survival of endothelial cells and pericytes through upregulated expression of vascular endothelial growth factor A (VEGFA), VEGF receptor 2, platelet-derived growth factor-B (PDGFB), PDGF receptor β, and angiopoietin-1 after SCI. Taken together, this study provides evidence for the mechanisms underlying the promotion of microcirculation reconstruction after SCI by microtubule stabilization treatment. Importantly, this study suggests the potential of microtubule stabilization as a therapeutic target to reduce microcirculation dysfunction after SCI in the clinic.

Salvianolic Acid Alleviated Blood-Brain Barrier Permeability in Spontaneously Hypertensive Rats by Inhibiting Apoptosis in Pericytes via P53 and the Ras/Raf/MEK/ERK Pathway.

ObjectiveTo investigate the effect of salvianolic acid A (SA) on the permeability of blood–brain barrier (BBB) and brain microvascular pericyte apoptosis in spontaneously hypertensive rats (SHR).MethodsEvans Blue was used to determine the BBB permeability in control rats and SHR. Western blotting was used to evaluate the expression levels of relevant proteins in the pericytes isolated from the differentially treated animals. An in vitro model of hypertension was established by stimulating pericytes with angiopoietin-2 (Ang2). MTT assay was used to assess cell viability, and apoptosis and cell cycle distribution were analyzed by flow cytometry.ResultsSA attenuated BBB permeability in SHR in a dose-dependent manner. It downregulated pro-apoptotic proteins including p53, p21, Fas, FasL, cleaved-caspase 3/caspase 3 and Bax in the pericytes of SHR and upregulated CDK6, cyclin D1, CDK2, cyclin E and Bcl2. In addition, SA activated the Ras/Raf/MEK/ERK pathway in a dose-dependent manner by increasing the levels of Ras, Raf, p-MEK1, p-MEK2, p-ERK1 and p-ERK2. Finally, SA reduced Ang2-induced apoptosis of cerebral microvessels pericytes and decreased the proportion of cells in the G0/G1 phase of the cell cycle by inhibiting the p53 pathway and activating the Ras/Raf/MEK/ERK pathway.ConclusionSA reduced BBB permeability in spontaneously hypertensive rats, possibly by inhibiting Ang2-induced apoptosis of pericytes by activating the Ras/Raf/MEK/ERK pathway.

A TGFβ1‐BIGH3‐Apoptosis Axis Comprising Peptidase and Integrin α3β1 Promotes Renal Cell Death

Transforming growth factor‐β1 (TGFβ1) helps contribute to diabetes complications in various organs including the renal system. TGFβ1 signaling activates genes encoding various extracellular matrix (ECM) molecules that accumulate and promote renal cell injury, increasing the probability of end stage renal disease (ESRD). The mechanisms underlying development of ESRD are complex and not known completely. We have shown previously that TGFβ1 strongly stimulates renal proximal tubule epithelial (PTE) cells to express the gene that encodes an ECM protein called BIGH3. This newly‐synthesized endogenous BIGH3 protein increased PTE cell apoptosis. Treating PTE cells with exogenous recombinant BIGH3 protein increased apoptosis. Anti‐BIGH3 antibody blocked endogenous and exogenous BIGH3‐mediated apoptosis. These findings signify a TGFβ1‐BIGH3‐Apoptosis axis that augments kidney disease progression. BIGH3’s unstructured C‐terminus encodes integrin‐binding sequences EPDIM and RGD. In the kidney, BIGH3 C‐terminal proteolysis (CTP) provides bioactive C‐terminal fragments (BCTF) that comprise EPDIM and RGD and account for PTE cell apoptosis. The EPDIM motif in BIGH3 induced two‐fold greater apoptosis of PTE cells when compared to apoptosis induced by the RGD motif. In order to provide evidence that BIGH3 CTP and integrin interaction with EPDIM is required for PTE cell apoptosis, we used recombinant BIGH3, PTE cells, BIGH3 mutagenesis, protease inhibitors, TUNEL, immunoblots, integrin α3 antibody and siRNA. Homology modeling and molecular docking software Raptor X, ZDOCK and AutoDock Vina were used to generate monomeric and heterodimer models of α3β1 integrin; followed by in silico analysis of EPDIM interaction with α‐integrin subunit. Results show that a serine peptidase accomplishes BIGH3 CTP, which we regard as mechanistic in the proposed axis. A 21% chemical inhibition of BIGH3 CTP resulted in a 43% decrease in PTE cell apoptosis. In silico analysis indicates that the BCTF encoding EPDIM, bound to α3 subunit, prevents full activation and disrupts integrin‐mediated pro‐survival signals. We conclude that BIGH3 mediates at least some of the destructive arm of TGFβ1 signaling in diabetic nephropathy. BIGH3 CTP provides local mediator BCTFs that act through paracrine or autocrine pathways to block α3β1 pro‐survival signaling leading to PTE cell apoptosis. The data indicate that molecules of the axis pathway, including BIGH3, BIGH3 peptidase, and BIGH3 BCTFs are potential therapeutic targets to treat diabetic nephropathy. In a broader perspective, we have evidence that a similar mechanism is active in retinal endothelial and retinal pericyte apoptosis in diabetic complications of the ocular system.

DJ-1 protects retinal pericytes against high glucose-induced oxidative stress through the Nrf2 signaling pathway

Oxidative stress has been associated with the etipathogenesis of Diabetic retinopathy (DR). Studies have shown that DJ-1 plays an important role in regulating the reactive oxygen species (ROS) production and resistance to oxidative stress-induced apoptosis. This study aimed to investigate whether DJ-1 upregulates oxidative stress and prevents damage to retinal capillary pericytes by increasing antioxidant capacity through the Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Nrf2 is a redox-sensitive transcription factor that encode antioxidant enzymes and phase II metabolic enzymes, activation of Nrf2 functions is one of the critical defensive mechanisms against oxidative stress in many tissues. Our results showed after DJ-1 overexpression, apoptosis of rat retinal pericytes (RRPs) decreased, the ratio of B-cell lymphoma-2 (Bcl-2) to BCL2-Associated X Protein (BAX) increased, the production of ROS decreased, and the protein expression and activity of manganese superoxide dismutase (MnSOD, also called SOD2) and catalase (CAT) increased. DJ-1 overexpression activated Nrf2 expression, however, after Nrf2 silencing, apoptosis of RRPs increased, the ratio of Bcl-2 to BAX decreased, the production of ROS increased, the protein expression of MnSOD and CAT decreased, and the expression of heme oxygenase-1 (HO-1), NADP(H) quinone oxidoreductase (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) decreased. These data suggest that enhancement of the Nrf2 pathway is a potential protective strategy for the treatment of DR. Therefore, DJ-1 may prevent high glucose-induced oxidative stress and RRPs apoptosis through the Nrf2 signaling pathway, thereby preventing the early onset and progression of DR.

Identification of early pericyte loss and vascular amyloidosis in Alzheimer\u2019s disease retina

Pericyte loss and deficient vascular platelet-derived growth factor receptor-β (PDGFRβ) signaling are prominent features of the blood–brain barrier breakdown described in Alzheimer’s disease (AD) that can predict cognitive decline yet have never been studied in the retina. Recent reports using noninvasive retinal amyloid imaging, optical coherence tomography angiography, and histological examinations support the existence of vascular-structural abnormalities and vascular amyloid β-protein (Aβ) deposits in retinas of AD patients. However, the cellular and molecular mechanisms of such retinal vascular pathology were not previously explored. Here, by modifying a method of enzymatically clearing non-vascular retinal tissue and fluorescent immunolabeling of the isolated blood vessel network, we identified substantial pericyte loss together with significant Aβ deposition in retinal microvasculature and pericytes in AD. Evaluation of postmortem retinas from a cohort of 56 human donors revealed an early and progressive decrease in vascular PDGFRβ in mild cognitive impairment (MCI) and AD compared to cognitively normal controls. Retinal PDGFRβ loss significantly associated with increased retinal vascular Aβ40 and Aβ42 burden. Decreased vascular LRP-1 and early apoptosis of pericytes in AD retina were also detected. Mapping of PDGFRβ and Aβ40 levels in pre-defined retinal subregions indicated that certain geometrical and cellular layers are more susceptible to AD pathology. Further, correlations were identified between retinal vascular abnormalities and cerebral Aβ burden, cerebral amyloid angiopathy (CAA), and clinical status. Overall, the identification of pericyte and PDGFRβ loss accompanying increased vascular amyloidosis in Alzheimer’s retina implies compromised blood–retinal barrier integrity and provides new targets for AD diagnosis and therapy.

Natriuretic Peptides Attenuate Retinal Pathological Neovascularization Via Cyclic Guanosine Monophosphate Signaling in Pericytes and Astrocytes.

In proliferative retinopathies, complications derived from neovascularization cause blindness. During early disease, pericyte's apoptosis contributes to endothelial dysfunction and leakage. Hypoxia then drives VEGF (vascular endothelial growth factor) secretion and pathological neoangiogenesis. Cardiac ANP (atrial natriuretic peptide) contributes to systemic microcirculatory homeostasis. ANP is also formed in the retina, with unclear functions. Here, we characterized whether endogenously formed ANP regulates retinal (neo)angiogenesis. Approach and Results: Retinal vascular development and ischemia-driven neovascularization were studied in mice with global deletion of GC-A (guanylyl cyclase-A), the cGMP (cyclic guanosine monophosphate)-forming ANP receptor. Mice with a floxed GC-A gene were interbred with Tie2-Cre, GFAP-Cre, or PDGF-Rβ-CreERT2 lines to dissect the endothelial, astrocyte versus pericyte-mediated actions of ANP in vivo. In neonates with global GC-A deletion (KO), vascular development was mildly delayed. Moreover, such KO mice showed augmented vascular regression and exacerbated ischemia-driven neovascularization in the model of oxygen-induced retinopathy. Notably, absence of GC-A in endothelial cells did not impact retinal vascular development or pathological neovascularization. In vitro ANP/GC-A/cGMP signaling, via activation of cGMP-dependent protein kinase I, inhibited hypoxia-driven astrocyte's VEGF secretion and TGF-β (transforming growth factor beta)-induced pericyte apoptosis. In neonates lacking ANP/GC-A signaling in astrocytes, vascular development and hyperoxia-driven vascular regression were unaltered; ischemia-induced neovascularization was modestly increased. Remarkably, inactivation of GC-A in pericytes retarded physiological retinal vascularization and markedly enhanced cell apoptosis, vascular regression, and subsequent neovascularization in oxygen-induced retinopathy. Protective pericyte effects of the ANP/GC-A/cGMP pathway counterregulate the initiation and progression of experimental proliferative retinopathy. Our observations indicate augmentation of endogenous pericyte ANP signaling as target for treatment of retinopathies associated with neovascularization.

Endothelial specific deletion of FOXO1 alters pericyte coverage in the developing retina

Pericytes are mural cells that cover small blood vessels. While defects in pericyte coverage are known to be involved in various vessel related pathologies, including diabetic retinopathy, the molecular mechanisms underlying pericyte coverage are not fully understood. In this study, we investigated the contribution of the forkhead transcription factor FOXO1 in endothelial cells to pericyte coverage in the developing retina. We observed retinal pericytes in tamoxifen-inducible endothelium-specific Foxo1 deletion mice. Tamoxifen was injected at postnatal day 1–3 and the retinas were harvested at P21. Our results demonstrated that Foxo1 deletion in the endothelium affected arteriole pericyte morphology without altering pericyte number, proliferation, and apoptosis. We hypothesized that abnormal pericyte morphogenesis in the knockout retina was caused by impaired pericyte differentiation. FOXO1 silencing by siRNA in the primary artery endothelium further revealed that THBS1 (thrombospondin 1), which promotes pericyte differentiation via TGFβ activation, was reduced in the FOXO1-deficient endothelium. Immunohistochemistry of FOXO1 knockout mice showed reduced numbers of phospho-Smad3+ arteriole pericytes compared with wild-type mice. In addition, endothelium-pericyte co-culture analysis revealed that pericytes cultured with FOXO1-deficient endothelial cells failed to differentiate sufficiently; this failure was partially rescued by the addition of recombinant THBS1 to the supernatant.The findings suggest that endothelial FOXO1 contributes to pericyte differentiation via regulation of THBS1 expression. This study provides new insights into the molecular mechanism of pericyte coverage in the context of endothelium-derived regulation and highlights a new therapeutic target for pericyte-related pathology.

DJ-1/PARK7 inhibits high glucose-induced oxidative stress to prevent retinal pericyte apoptosis via the PI3K/AKT/mTOR signaling pathway

Reactive oxygen species (ROS) act through multiple pathways to induce apoptosis of retinal capillary pericytes, which is an early marker and the primary cause of the progression of diabetic retinopathy. However, the specific molecular mechanisms behind ROS-induced retinal capillary pericyte loss in diabetic retinopathy remains elusive. In this study, we investigated the molecular regulation and effects of DJ-1/PARK7 on oxidative stress and injury of rat retinal pericytes (RRPs). To perform the research, RRPs were isolated from rat retina and cultured in medium with for 2 days: control group (5.6 mM glucose), high glucose group (30 mM glucose), hypertonic group (5.6 mM glucose + 24.4 mM mannitol). We found decreased expression of DJ-1 and increased apoptosis of RRPs in high glucose group. To further study the role of DJ-1, four groups were divided as follows: normal control group (5.6 mM glucose), high glucose (30 mM glucose), empty vector control group (pcDNA3.1,30 mM glucose), DJ-1 overexpression group (pcDNA3.1-myc-DJ-1,30 mM glucose). DJ-1, P53, p-P53, cleaved caspase-3, manganese superoxide dismutase (MnSOD), catalase (CAT) and PI3K/Akt/mTOR signaling pathway in each group was detected by Western Blot. RRPs apoptosis was detected by Terminal-deoxynucleoitidyl Transferase mediated Nick End Labeling (TUNEL) and 4′6- diamidino-2-phenylindole (DAPI). Mitochondrial function was detected by jc-1 and fluorescent probes DCFH-DA was used to determine reactive oxygen species (ROS). We found that high glucose (30 mM) lasting two days can induce significant apoptosis of RRPs, increase ROS production and expressions of p-p53 and active caspase-3, impair mitochondrial function, decrease the activities of MnSOD and CAT, and decrease expression of DJ-1, p-AKT and p-mTOR. In contrast, DJ-1/PARK7 overexpression significantly increases expression of DJ-1, p-AKT and p-mTOR, increases expression and activities of MnSOD and CAT, improves mitochondrial function, decreases expression of apoptotic gene protein p-p53 and active caspase-3, reduces ROS production and reduces the apoptotic rate of RRPs induced by high glucose. These results suggest that DJ-1 may play a role in protecting RRPs from high glucose induced-oxidative injury. DJ-1 might improve mitochondrial function, inhibit ROS production and enhance antioxidant capacity to reduce apoptosis of retinal pericytes through the PI3K/AKT/mTOR signaling pathway which may be related to early pathogenesis of diabetic retinopathy.

Asymmetric dimethylarginine aggravates blood-retinal barrier breakdown of diabetic retinopathy via inhibition of intercellular communication in retinal pericytes.

Blood-retinal barrier breakdown is the main pathological characteristics of diabetic retinopathy (DR). Asymmetric dimethylarginine (ADMA) was reported to be elevated in DR patients. In this study, we observed the dynamic profile of ADMA, retinal morphology and permeability of BRB at 2, 4 or 8week of diabetic rats induced by a single intraperitoneal injection of streptozocin (60mg/kg) and in cultured rat retinal pericytes pretreated with D-glucose (30mM) for 1, 3, 5 and 7days or ADMA (3, 10, 30 μM) for 24, 48 and 72h, trying to explore the effects of ADMA on blood-retinal barrier in DR. Gap junction intercellular communication (GJIC) and the expression of blood-retinal barrier-specific component connexin 43 (Cx43) were examined in diabetic rats or cultured retinal pericytes to elucidate whether ADMA impacted blood-retinal barrier function via damaging Cx43-GJIC. The results showed that with increasing duration of diabetes, the ultrastructure of blood-retinal barrier of diabetic rats appeared cell junction damage, apoptosis of retinal pericytes and breakdown of barrier successively. The increases in retinal permeability, ADMA levels and Cx43 expression, and abnormal GJIC were observed in diabetic rats and retinal pericytes exposed to D-glucose (30mM). A glucose-like effect was seen using ADMA or another L-arginine analogue NG-monomethyl-L-arginine or dimethylarginine dimethylaminohydrolases (DDAHs) siRNA, implicating that ADMA aggravated the breakdown of blood-retinal barrier via damaging Cx43-GJIC.