Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/TIE2 interaction during sepsis is a potential therapeutic target.
This was an investigation of whether mitochondrial dysfunction plays a role in pericyte loss, a prominent lesion of diabetic retinopathy. The findings indicate that in a high-glucose condition, mitochondria of retinal pericytes display significant fragmentation, metabolic dysfunction, and reduced extracellular acidification. These altered mitochondrial characteristics could play a role in the accelerated apoptosis associated with the retinal pericytes in diabetic retinopathy.
Mitochondrial dysfunction is known to play a role in retinal vascular cell loss, a prominent lesion of diabetic retinopathy. High glucose (HG) has been reported to induce mitochondrial fragmentation and dysfunction in retinal endothelial cells, contributing to apoptosis. In this study, the effects of HG on mitochondrial morphology, membrane potential, and metabolic changes and whether they could contribute to HG-induced apoptosis in retinal pericytes were investigated.
Bovine retinal pericytes (BRPs) were grown in normal or HG medium for 7 days. Both sets of cells were double stained with mitochondrial membrane potential–independent dye and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) and imaged by confocal microscopy. The images were analyzed for average mitochondria shape, by using form factor and aspect ratio values, and membrane potential changes, by using the ratio between the red and green dye. BRPs grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification with a flux analyzer and apoptosis by TUNEL assay.
BRPs grown in HG media exhibited significant fragmentation of mitochondria and increased membrane potential heterogeneity compared with the BRPs grown in normal medium. Concomitantly, BRPs grown in HG showed reduced steady state and maximum oxygen consumption and reduced extracellular acidification. Number of TUNEL-positive pericytes was increased in HG condition as well.
In HG condition, mitochondria of retinal pericytes display significant fragmentation, metabolic dysfunction, and reduced extracellular acidification. The detrimental effects of HG on mitochondrial function and cellular metabolism could play a role in the accelerated apoptosis associated with the retinal pericytes in diabetic retinopathy.
Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy.
We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection).
ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-β1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.
The role of retinal pericytes in diabetic retinopathy was not clear. This study demonstrated a novel role of retinal pericytes, suggesting that their immunoregulatory activity could contribute to the pathogenesis of diabetic retinopathy.
To test the hypothesis that retinal pericytes (RPCs) are immunosuppressive; therefore, their loss of function under hyperglycemic conditions favors retinal inflammation and contributes to the pathogenesis of diabetic retinopathy (DR).
Isolated mouse and human RPCs were tested in T cell function assays to evaluate their capability of inhibiting T cell responses. To elucidate the underlying mechanisms, transwell systems, blocking mAbs against PD-L1 and IL-10 were used. The efficacy of RPCs in protecting retinal endothelial cells (RECs) from inflammation-induced apoptosis was assessed by apoptosis detection staining. Finally, to test whether hyperglycemic conditions impair the immunomodulatory activity of RPCs, RPCs pre-incubated in high glucose or methylglyoxal (MGO) were evaluated using the T cell proliferation assays.
RPCs profoundly inhibited activated T cell proliferation and inflammatory cytokine production. The T cell inhibitory activity of RPCs was decreased, but was not abolished, in transwell experiments. RPCs express PD-L1, and blocking PD-L1 reduced RPCs' efficacy of T cell inhibition. RPCs also produce IL-10, and neutralization of IL-10 reduced their immunosuppressive activity. There were significantly reduced numbers of inflammation-induced apoptosis-detected RECs in the presence of RPCs. Incubation of RPCs with either high glucose or MGO reduced the activity of RPCs to inhibit activated T cell proliferation.
RPCs are highly immunosuppressive and they protected RECs from inflammation-mediated apoptosis. Hyperglycemic conditions impaired the T cell inhibitory activity of RPCs. These results reveal a new function of RPCs, and its regulation under hyperglycemic conditions. This may represent a novel mechanism by which RPCs contribute to preservation of retinal integrity in diseases, including DR.
To test the hypothesis that autoantibodies against retinal pericytes could develop in diabetic retinopathy, and that these autoantibodies could induce retinal pericyte dysfunction/death via complement.
Human primary retinal pericytes cultured in media containing normal (5 mM) or high (30 mM) glucose concentrations were incubated with normal human sera in the presence of a retinal pericyte-reactive antibody, then their viability was assessed by a BCECF-based cytotoxicity assay, and their function was assessed by a T-cell proliferation assay. The pericytes were also analyzed by RT-PCR and flow cytometry to detect CD38, an established diabetes-associated cell surface autoantigen. The potential of the anti-CD38 antibodies in inducing pericyte cellular injury was evaluated using the same cytotoxicity assays. In addition, autoantibody-mediated cytotoxicity in mouse retinal pericytes sensitized by sera from mice with developing diabetic retinopathy or control normal mice were also studied.
Retinal pericyte–reactive antibodies induced cellular damage by activating complement in the serum. The antibody-injured pericytes had reduced efficacy in inhibiting T cells. Hyperglycemic culture conditions rendered pericytes more susceptible to antibody-mediated attack. CD38 was expressed in retinal pericytes, and upregulated by TNF-α and IFN-γ, and anti-CD38 antibodies induced pericyte cytotoxicity. Retinal pericytes sensitized with sera from chronic diabetic mice suffered significantly augmented cytotoxicity compared with those sensitized with sera from the control mice.
The autoantibody-initiated complement activation could be a mechanism underlying the loss of function, and eventually, death of retinal pericytes in diabetic patients, suggesting that inhibiting complement activation could be a novel therapeutic approach.
Data presented in this report suggest that autoantibodies against retinal pericyte cell surface antigens induce pericyte cytotoxicity through complement, which could contribute to the development of diabetic retinopathy.
Pericyte loss is a cardinal feature of early diabetic retinopathy. We previously reported that highly oxidized-glycated low density lipoprotein (HOG-LDL) induces pericyte apoptosis in vitro. In this study, we investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathways in HOG-LDL-induced apoptosis in human pericytes.
Human retinal capillary pericytes (HRCP) were exposed to native LDL (N-LDL) and HOG-LDL, and apoptosis was measured using flow cytometry. Time- and dose-dependent responses of extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) following exposure to N-LDL or HOG-LDL were determined using western blotting. U0126 (ERK inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) were used to determine the role of MAPK signaling in HOG-LDL-induced apoptosis.
HOG-LDL induced apoptosis in HRCP in a dose-dependent manner at concentrations from 5 to 50 mg/l, with a constant effect from 50 to 200 mg/l. When compared to serum-free medium (SFM), this effect of HOG-LDL was found to be significant at all doses above 10 mg/l. In contrast, N-LDL at 200 mg/l did not induce apoptosis compared with SFM. Exposure to N-LDL versus HOG-LDL induced similar phosphorylation of ERK, p38, and JNK, peaking at 5 min, with similar dose-dependent responses up to 25 mg/l that were constant from 25 to 100 mg/l. Blocking of the ERK, p38, and JNK pathways did not inhibit pericyte apoptosis induced by HOG-LDL.
Our data suggest that apoptosis induced by HOG-LDL in HRCP is independent of the activation of MAPK signaling pathways.
Kidney pericytes were recently identified as collagen-Iα1 producing cells in healthy kidney, but the developmental, physiological and pathological roles of kidney pericytes remain poorly understood. Pericytes are stromal-derived cells that envelop, and have intimate connections with adjacent capillary endothelial cells (ECs). Recent studies in eye and brain have revealed that pericytes are crucial for angiogenesis, vascular stability and vessel integrity.In response to kidney injury, pericytes promptly migrate away from the capillary wall into the interstitial space. Here, pericytes are activated and differentiate into scar-forming myofibroblasts. In the absence of pericytes, peritubular capillaries are destabilized leading to vascular regression. Consequently, capillary loss and fibrosis following kidney injury are intimately linked and hinge centrally around pericyte detachment from ECs.Kinetic mathematical modeling demonstrated that pericytes are the major source of myofibroblasts in fibrotic kidney. Comprehensive genetic fate mapping studies of nephron epithelia or kidney stroma has demonstrated that epithelial cells do not migrate outside of epithelial compartment to become myofibroblasts rather that interstitial pericytes are progenitors of scar-forming myofibroblasts. Bidirectional signaling between pericytes and ECs is necessary for pericyte detachment from peritubular capillaries.In the present review, we summarize the pathologically vital roles of kidney pericytes in fibrosis including our new findings. The study of kidney pericytes and endothelial-pericyte crosstalk will identify novel therapeutic targets for currently incurable chronic kidney diseases.
capillary rarefaction; chronic kidney diseases; endothelial cells; epithelial-to-mesenchymal transition (EMT); fibrosis; kidney pericytes; kidney injury; myofibroblasts; peritubular capillary
This is the first report on connexin 43, a principal gap junction protein that is downregulated by diabetes in the retina. The effect of Cx43 downregulation in the diabetic retina could result in reduced cell-to-cell communication and apoptosis in retinal vascular cells, leading to acellular capillaries and pericyte loss.
To examine whether diabetes-induced connexin 43 downregulation promotes retinal vascular lesions characteristic of diabetic retinopathy (DR).
Two animal models, streptozotocin-induced diabetic mice and Cx43 heterozygous knockout (Cx43±) mice, were studied to directly assess whether diabetes reduces the expression of retinal Cx43, which, in turn, contributes to retinal vascular cell loss by apoptosis. Retinal Cx43 protein levels were assessed in nondiabetic control mice, diabetic mice, and Cx43± mice by Western blot analysis, and Cx43 localization and distribution in the retinal vascular cells were studied by immunostaining of retinal trypsin digests (RTDs). In parallel, RTDs were stained with hematoxylin and periodic acid Schiff to determine pericyte loss (PL) and acellular capillaries (AC), and TUNEL assays were performed to determine retinal vascular cell apoptosis.
Western blot analysis indicated significant reductions in retinal Cx43 protein levels in diabetic mice and Cx43± mice compared with those of nondiabetic mice. Similarly, a significant reduction in Cx43 immunostaining was observed in the retinal capillaries of diabetic mice and Cx43± mice compared with those of control mice. Both diabetic and age-matched Cx43± mice exhibited increased amount of PL, AC, and TUNEL-positive cells compared with control mice.
Diabetes-induced inhibition of Cx43 expression contributes to vascular cell apoptosis in retinas of diabetic mice. This suggests that reduced Cx43 expression plays a critical role in the development of AC and PL associated with DR.
Regulators of G-protein signaling (RGS) are involved in a wide variety of functions, including olfaction, vision, and cell migration. RGS5 has a perivascular expression pattern and was recently identified as a marker for brain pericytes. This suggests a role for RGS5 in vascular development and pericyte biology. We have created a mouse line which lacks the rgs5 gene and replaced it with a green fluorescent protein (GFP) reporter (rgs5GFP/GFP). The mice are viable and fertile and display no obvious developmental defects, and the vasculature appears to develop normally with proper pericyte coverage. Also, no differences were observed in the vasculature under pathological conditions, such as tumor growth and oxygen-induced retinopathy. The GFP expression in pericytes of rgs5GFP mice allows detection and sorting of these cells, thereby providing a valuable novel tool for pericyte research.
The blood–spinal cord barrier (BSCB) regulates molecular exchange between blood and spinal cord. Pericytes are presumed to be important cellular constituents of the BSCB. However, the regional abundance and vascular functions of spinal cord pericytes have yet to be determined. Utilizing wild-type mice, we show that spinal cord pericyte capillary coverage and number compared with the brain regions are reduced most prominently in the anterior horn. Regional pericyte variations are highly correlated with: (1) increased capillary permeability to 350 Da, 40,000 Da, and 150,000 Da, but not 2,000,000 Da fluorescent vascular tracers in cervical, thoracic, and lumbar regions and (2) diminished endothelial zonula occludens-1 (ZO-1) and occludin tight junction protein expression. Pericyte-deficient mutations (PdgfrβF7/F7 mice) resulted in additional pericyte reductions in spinal cord capillaries leading to overt BSCB disruption to serum proteins, accumulation in motor neurons of cyotoxic thrombin and fibrin and motor neuron loss. Barrier disruption in perciyte-deficient mice coincided with further reductions in ZO-1 and occludin. These data suggest that pericytes contribute to proper function of the BSCB at the capillary level. Regional reductions in spinal cord pericytes may provide a cellular basis for heightened spinal cord barrier capillary permeability and motor neuron loss.
blood–brain barrier; capillaries; endothelium; pericytes; vascular biology
Oxidized and/or glycated LDL may mediate capillary injury in diabetic retinopathy. The mechanisms may involve pro-inflammatory and pro-oxidant effects on retinal capillary pericytes. In this study, these effects, and the protective effects of pigment epithelium-derived factor (PEDF), were defined in a primary human pericyte model. Human retinal pericytes were exposed to 100 µg/ml of native LDL (N-LDL) or heavily oxidized glycated LDL (HOG-LDL) with or without PEDF at 10–160 nM for 24 h. To assess pro-inflammatory effects, monocyte chemoattractant protein-1 (MCP-1) secretion was measured by ELISA, and nuclear factor kappa B (NF-κB) activation was detected by immunocytochemistry. Oxidative stress was determined by measuring intracellular reactive oxygen species (ROS), peroxynitrite (ONOO−) formation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. The results showed that MCP-1 was significantly increased by HOG-LDL, and the effect was attenuated by PEDF in a dose-dependent manner. PEDF also attenuated the HOG-LDL-induced NF-κB activation, suggesting that the inhibitory effect of PEDF on MCP-1 was at least partially through the blockade of NF-κB activation. Further studies demonstrated that HOG-LDL, but not N-LDL, significantly increased ONOO− formation, NO production, and iNOS expression. These changes were also alleviated by PEDF. Moreover, PEDF significantly ameliorated HOG-LDL-induced ROS generation through up-regulation of SOD1 expression. Taken together, these results demonstrate pro-inflammatory and pro-oxidant effects of HOG-LDL on retinal pericytes, which were effectively ameliorated by PEDF. Suppressing MCP-1 production and thus inhibiting macrophage recruitment may represent a new mechanism for the salutary effect of PEDF in diabetic retinopathy and warrants more studies in future.
PEDF; oxidized LDL; inflammation; oxidative stress
An early and significant event in diabetic retinopathy is the loss of retinal microvascular pericytes. Studies were performed to investigate pathways through which an advanced glycation endproduct and tumor necrosis factor (TNF)-α stimulate apoptosis in retinal pericytes through the activation of the pro-apoptotic transcription factor Forkhead box O1 (FOXO1).
Human retinal pericytes were stimulated by carboxymethyllysine (CML)-collagen, an advanced glycation endproduct, or TNF-α in vitro. Apoptosis was assessed by measuring cytoplasmic histone-associated DNA. The role of FOXO1 was examined by RNA interference (RNAi), and specific inhibitors were used to investigate the role of p38 and Jun N-terminal kinase mitogen-activated protein kinase (JNK MAP) kinases, Akt, and nuclear factor kappa B (NF-κB). Caspase-3 activity was measured with a luminescent substrate, and FOXO1 DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA).
TNF-α and CML-collagen but not control collagen stimulated apoptosis, caspase-3 activity, and FOXO1 DNA-binding activity in pericytes. Silencing FOXO1 by small interfering RNA prevented apoptosis of pericytes in response to both TNF-α and CML-collagen. By use of specific inhibitors, we demonstrated that both FOXO1 activation and subsequent apoptosis was mediated, in part, by p38 and JNK MAP kinases. In contrast Akt and NF-κB inhibitors had the opposite effect on pericyte apoptosis.
The results demonstrate pathways through which two different mediators, TNF-α and an advanced glycation endproduct, can induce pericyte apoptosis through activation of the transcription factor FOXO1.
In diabetic retinopathy, collapse of the retinal vasculature is associated with loss of the pericytes. These are contractile cells that together with endothelial cells form the terminal arterioles of the retina. The cause of the loss of pericytes is not known. Recently, it has been discovered that type 1 diabetes is caused by forbidden clones of cytotoxic T lymphocytes, which destroy the insulin-making cells with exquisite specificity. In the light of this, I postulate that an antigenically-related forbidden clone of cytotoxic T lymphocytes selectively destroys the pericytes and that this is the cause of the vascular collapse of diabetic retinopathy. If this is so, the therapeutic implications are immense, involving a switch from ineffectual tight glycemic control to immunotherapy. This is already used as immunosuppression to prevent organ transplant rejection, and as the immune ablation and autologous bone marrow cell reconstitution that has saved the lives of patients with lethally-severe scleroderma. Once the pericyte surface auto-antigen for the T lymphocytes has been isolated, selective destruction of the pathogenic T lymphocytes would be possible by manufacture and use of cytotoxic auto-antigen complexes, which arrests progression of the retinopathy.
pericytes; diabetic retinopathy; autoimmunity; T cell forbidden clones; immunotherapy
One of the histopathologic hallmarks of early diabetic retinopathy is the loss of pericytes. Evidences suggest that the pericyte loss in vivo is mediated by apoptosis. However, the underlying cause of pericyte apoptosis is not fully understood. This study investigated the influence of methylglyoxal (MGO), a reactive α-dicarbonyl compound of glucose metabolism, on apoptotic cell death in bovine retinal pericytes. Analysis of internucleosomal DNA fragmentation by ELISA showed that MGO (200 to 800 µM) induced apoptosis in a concentration-dependent manner. Intracellular reactive oxygen species were generated earlier and the antioxidant, N-acetyl cysteine, inhibited the MGO-induced apoptosis. NF-κB activation and increased caspase-3 activity were detected. Apoptosis was also inhibited by the caspase-3 inhibitor, Z-DEVD-fmk, or the NF-κB inhibitor, pyrrolidine dithiocarbamate. These data suggest that elevated MGO levels observed in diabetes may cause apoptosis in bovine retinal pericytes through an oxidative stress mechanism and suggests that the nuclear activation of NF-κB are involved in the apoptotic process.
Retina; Pericytes; Methylglyoxal; Pyruvaldehyde; Apoptosis; Reactive Oxygen Species; NF-kappaB
Cellular apoptosis induced by hyperglycemia occurs in many vascular cells and is critical to initiate diabetic pathologies. In the retina, pericyte apoptosis, the most specific vascular pathology attributed to hyperglycemia, is linked to the loss of PDGF actions due to unknown mechanisms. Our study demonstrated that hyperglycemia persistently activated PKCδ and p38α MAPK to increase the expression of a novel target, SHP-1, leading to PDGF receptor-β dephosphorylation and actions, and increased pericyte apoptosis, independent of NF-κB. These findings were also observed in diabetic mouse retinas, which were not reversed by achieving normoglycemia with insulin. Unlike diabetic controls, diabetic Prkcd−/− mice did not exhibit p38α MAPK/SHP-1 activation, PDGF resistance or acellular capillaries. Since PKCδ/p38α MAPK/SHP-1 activation are also induced in the brain pericytes and renal cortex by diabetes, these findings have elucidated a new pathway by which hyperglycemia can induce PDGF resistance and increase vascular cell apoptosis to cause diabetic vascular complications.
Advanced glycation end products (AGEs) and the renin-angiotensin system (RAS) are both implicated in the development of diabetic retinopathy. How these pathways interact to promote retinal vasculopathy is not fully understood. Glyoxalase-I (GLO-I) is an enzyme critical for the detoxification of AGEs and retinal vascular cell survival. We hypothesized that, in retina, angiotensin II (Ang II) downregulates GLO-I, which leads to an increase in methylglyoxal-AGE formation. The angiotensin type 1 receptor blocker, candesartan, rectifies this imbalance and protects against retinal vasculopathy.
RESEARCH DESIGN AND METHODS
Cultured bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRP) were incubated with Ang II (100 nmol/l) or Ang II+candesartan (1 μmol/l). Transgenic Ren-2 rats that overexpress the RAS were randomized to be nondiabetic, diabetic, or diabetic+candesartan (5 mg/kg/day) and studied over 20 weeks. Comparisons were made with diabetic Sprague-Dawley rats.
In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO•), the latter being a known negative regulator of GLO-I in BRP. In BREC and BRP, candesartan restored GLO-I and reduced NO•. Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I. In diabetic Ren-2 rats, candesartan reduced retinal acellular capillaries, inflammation, and inducible nitric oxide synthase and NO•, and restored GLO-I.
We have identified a novel mechanism by which candesartan improves diabetic retinopathy through the restoration of GLO-I.
The two most widely investigated animal models for diabetic retinopathy (DR) are the rat and dog. In dogs, aldose reductase (AR) is present only in retinal capillary pericytes and their destruction has been linked to polyol accumulation and resulting apoptosis. Since both rat capillary pericytes and endothelial cells have been reported to contain AR, the role of polyol pathway activity in capillary cell destruction has been investigated in rat retinal capillary pericyte (TR-rPCT) and endothelial (TR-iBRB) cells.
TR-rPCT and TR-iBRB cell lines were recloned and their identities were reconfirmed by characteristic immunostaining. Cells were cultured up to 72 h in media containing 50 mM glucose or galactose with/without the AR inhibitors or a sorbitol dehydrogenase inhibitor (SDI) or with 30 mM 3-fluoro-3-deoxyglucose. Polyol levels were determined by HPLC or 19F-NMR. Apoptosis was detected with TUNEL/DAPI staining.
Smooth muscle actin is present only in pericytes while only endothelial cells stain for von Willebrand factor and accumulate acetylated low-density lipoprotein. AR is present in both cells but AR levels are lower in endothelial cells. Aldehyde reductase is also present in both cells. Cells cultured in 50 mM glucose or galactose show significant polyol accumulation in pericytes but endothelial cells show little accumulation of galactitol and no accumulation of sorbitol. Sorbitol accumulation in pericytes resulted in increased cellular permeability and increased TUNEL staining, which was reduced by AR inhibition.
Although both rat retinal pericytes and endothelial cells contain AR, sorbitol accumulation and TUNEL staining primarily occur in pericytes and are inhibited by AR inhibitors.
The purpose of this study was to investigate the effects of hyperglycemia, its fluctuations, and glucose starvation on the expression of glucose-regulated protein 78/binding immunoglobulin protein (GRP78/BiP), one of the most commonly used markers of endoplasmic reticulum stress, in rat capillary pericytes and endothelial cells cultured separately and together.
Conditionally immortalized rat retinal pericyte and endothelial cell lines were cultured in dishes coated with collagen type I in Dulbecco's modified Eagle's medium containing 5.5 mM glucose. For cocultures, pericytes and endothelial cells were seeded together on rat tail collagen type I–coated cell culture plates. After 24 h of initial culture, the medium was replaced with serum-free medium containing 0–100 mM glucose for periods of up to 72 h. GRP78/BiP, caspase-3, and nuclear factor-κB expression were investigated using western blots.
No significant increase in GRP78/BiP expression was observed when pericytes, endothelial cells, or cocultures were exposed to either 25, 50, or 100 mM glucose for 48 h compared with the control level of 5.5 mM glucose. Similarly, no change in expression of GRP78/BiP was observed when media glucose levels were reduced from either 5.5 or 25 to 1 mM. GRP78/BiP expression significantly increased when cells were cultured for 24 h in glucose-deprived medium. This was accompanied by a time-dependent increase in the expression of caspase-3 and nuclear factor-κB.
In diabetic retinopathy, hyperglycemia has been reported to induce apoptosis in retinal capillary vascular cells, but these studies suggest that the apoptosis is not linked to the expression of GRP78/BiP, one of the most commonly used markers of endoplasmic reticulum stress. However, GRP78/BiP-linked apoptosis may play a role in vascular changes associated with retinal ischemia/reperfusion.
The identification of microvascular pericytes in vitro relies principally on morphological characteristics and growth dynamics, as there is a paucity of immunochemical markers for these cells. Consequently, an attempt was made to identify mAb reagents that would aid in both the rapid identification and enrichment of retinal capillary pericytes in vascular cell cultures. A panel of mAbs raised by xenogeneic immunization of mice with various tissues was screened for immunoreactivity with dissociated cultures of bovine retinal capillary pericytes. Two antibodies from the panel (3G5 and HISL-8) were seen to react with pericytes by indirect immunofluorescence. The mAb 3G5 was selected for further study. mAb 3G5 did not react with dissociated cultures of smooth muscle cells, endothelial cells, or retinal pigmented endothelial cells. The pericyte 3G5 antigen was insensitive to the action of trypsin; therefore, mAb 3G5 was used to selectively purify pericytes from trypsinized mixed retinal cell cultures by flow cytometry. 3G5+ pericytes (representing 8% of cells in a mixed retinal cell culture) were enriched at least nine-fold to represent greater than 70% of cells. The mAb 3G5 stained retinal capillaries in vivo with a fluorescence distribution consistent with pericyte staining. The 3G5 antigen of cultured pericytes was found to be a glycolipid of mobility intermediate between ganglioside markers GM1 and GM2.
We assessed whether Angiopoietin-2 (Ang2), a Tie2 ligand and partial antagonist of Angiopoietin-1 (Ang1), is required for early vessel destabilization during postischemic angiogenesis, when combined with vascular growth factors.
In vitro, matrigel co-cultures assessed endothelial-cell tube formation and pericyte recruitment after stimulation of VEGF-A, Apelin (APLN), Ang1 with or without Ang2. In a murine hindlimb ischemia model, adeno-associated virus (rAAV, 3×1012 virusparticles) transduction of VEGF-A, APLN and Ang1 with or without Ang2 (continuous or early expression d0-3) was performed intramuscularly (d-14). Femoral artery ligation was performed at d0, followed by laser doppler perfusion meassurements (LDI) 7 and 14. At d7 (early timepoint) and d14 (late timepoint), histological analysis of capillary/muscle fiber ratio (CMF-R, PECAM-1) and pericyte/capillary ratio (PC-R, NG2) was performed.
In vitro, VEGF-A, APLN and Ang1 induced ring formation, but only APLN and Ang1 recruited pericytes. Ang2 did not affect tube formation by APLN, but reduced pericyte recruitment after APLN or Ang1 overexpression. In vivo, rAAV.VEGF-A did not alter LDI-perfusion at d14, consistent with an impaired PC-R despite a rise in CMF-R. rAAV.APLN improved perfusion at d14, with or without continuous Ang2, increasing CMF-R and PC-R. rAAV.Ang1 improved perfusion at d14, when combined with rAAV.Ang2 (d0-3), accompanied by an increased CMF-R and PC-R.
The combination of early vessel destabilization (Ang2 d0-3) and continuous Ang1 overexpression improves hindlimb perfusion, pointing to the importance of early vessel destabilization and subsequent vessel maturation for enhanced therapeutic neovascularization.
Pericytes play a key role in the development of cerebral microcirculation. The exact role of pericytes in the neurovascular unit in the adult brain and during brain aging remains, however, elusive. Using adult viable pericyte-deficient mice, we show that pericyte loss leads to brain vascular damage by two parallel pathways: (1) reduction in brain microcirculation causing diminished brain capillary perfusion, cerebral blood flow and cerebral blood flow responses to brain activation which ultimately mediates chronic perfusion stress and hypoxia, and (2) blood-brain barrier breakdown associated with brain accumulation of serum proteins and several vasculotoxic and/or neurotoxic macromolecules ultimately leading to secondary neuronal degenerative changes. We show that age-dependent vascular damage in pericyte-deficient mice precedes neuronal degenerative changes, learning and memory impairment and the neuroinflammatory response. Thus, pericytes control key neurovascular functions that are necessary for proper neuronal structure and function, and pericytes loss results in a progressive age-dependent vascular-mediated neurodegeneration.
We compared three rat strains to determine if different strains develop early-stage diabetic retinopathy or sensory neuropathy at different rates.
Sprague Dawley, Lewis, and Wistar rats were made diabetic with streptozotocin. Diabetic and nondiabetic animals had retinal vascular pathology measured at eight months of diabetes. The number of cells in the retinal ganglion cell layer (GCL), retinal function (using electroretinography [ERG]), and retinal levels of inducible nitric oxide synthase (iNOS), cyclooxygenase2 (COX2), and vascular endothelial growth factor (VEGF) were measured at four months of diabetes. Tactile allodynia was assessed in hind paws at two months of diabetes.
Diabetes of eight months’ duration resulted in a significant increase in retinal degenerate capillaries and pericyte ghosts in Lewis and Wistar rats, but not in Sprague Dawley rats. A significant loss of cells in the GCL occurred only in diabetic Lewis rats, whereas Wistar and Sprague Dawley rats showed little change. Diabetes-induced iNOS and VEGF were statistically significant in all strains. Cyclooxygenase 2 (COX2) was significantly elevated in the Sprague Dawley and Wistar strains. Lewis rats showed a similar trend, however, the results were not statistically significant. All strains tended to show diabetes-induced impairment of dark-adapted b-wave amplitude, but only Sprague Dawley and Lewis strains had a significant reduction in latency. All strains showed significant tactile allodynia in peripheral nerves.
At the durations studied, Lewis rats showed accelerated loss of both retinal capillaries and ganglion cells in diabetes, whereas diabetic Wistar rats showed degeneration of the capillaries without significant neurodegeneration, and Sprague Dawley rats showed neither lesion. Identification of strains that develop retinal lesions at different rates should be of value in investigating the pathogenesis of retinopathy.
IGF-1 has been associated with the pathogenesis of diabetic retinopathy, although its role is not fully understood. Here we show that normoglycemic/normoinsulinemic transgenic mice overexpressing IGF-1 in the retina developed most alterations seen in human diabetic eye disease. A paracrine effect of IGF-1 in the retina initiated vascular alterations that progressed from nonproliferative to proliferative retinopathy and retinal detachment. Eyes from 2-month-old transgenic mice showed loss of pericytes and thickening of basement membrane of retinal capillaries. In mice 6 months and older, venule dilatation, intraretinal microvascular abnormalities, and neovascularization of the retina and vitreous cavity were observed. Neovascularization was consistent with increased IGF-1 induction of VEGF expression in retinal glial cells. In addition, IGF-1 accumulated in aqueous humor, which may have caused rubeosis iridis and subsequently adhesions between the cornea and iris that hampered aqueous humor drainage and led to neovascular glaucoma. Furthermore, all transgenic mice developed cataracts. These findings suggest a role of IGF-1 in the development of ocular complications in long-term diabetes. Thus, these transgenic mice may be used to study the mechanisms that lead to diabetes eye disease and constitute an appropriate model in which to assay new therapies.
To investigate early events leading to microvascular cell loss in diabetic retinopathy.
RESEARCH DESIGN AND METHODS
FOXO1 was tested in vivo by DNA binding activity and by nuclear translocation in microvascular cells in retinal trypsin digests. In vivo studies were undertaken in STZ-induced diabetic rats and Zucker diabetic fatty rats using the tumor necrosis factor (TNF)-specific blocker, pegsunercept, or by inhibiting FOXO1 with RNAi. Microvascular cell apoptosis, formation of pericyte ghosts, and acellular capillaries were measured. Upstream and downstream effects of high-glucose–induced FOXO1 were tested on rat microvascular endothelial cells (RMECs) by small-interfering RNA (siRNA) in vitro.
DNA binding or nuclear translocation of FOXO1, which was reduced by TNF inhibition, was elevated in type 1 and type 2 diabetic retinas. Diabetes stimulated microvascular cell apoptosis; pericyte ghost and acellular capillary development was inhibited by FOXO1 siRNA. High glucose in vitro decreased FOXO1 phosphorylation and DNA binding activity and decreased Akt phosphorylation in RMECs. High-glucose–stimulated FOXO1 DNA binding activity was mediated through TNF-α and formation of reactive oxygen species (ROS), while inhibitors of TNF and ROS and FOXO1 siRNA reduced high-glucose–enhanced RMEC apoptosis. The caspase-3/7 activity and capacity of high glucose to increase mRNA levels of several genes that regulate RMEC activation and apoptosis were knocked down by FOXO1 siRNA.
FOXO1 plays an important role in rat retinal microvascular cell loss in type 1 and type 2 diabetic rats and can be linked to the effect of high glucose on FOXO1 activation.
Pericytes are a heterogeneous group of extensively branched cells located in microvessels where they make focal contacts with endothelium. Pericytes stabilize blood vessels, regulate vascular tone, synthesize matrix, participate in repair and serve as progenitor cells, among other functions. Recent work has highlighted the role of pericytes and pericyte-like cells in fibrosis, where chronic injury triggers pericyte proliferation and differentiation into collagen-secretory, contractile myofibroblasts with migration away from vessels, causing microvascular rarefaction. In this review I summarize the developmental origins of kidney pericytes and perivascular fibroblasts, discuss pericyte to myofibroblast transition in type I diabetic nephropathy and describe the regulation of pericyte differentiation into myofibroblasts as a therapeutic target for treatment of diabetic nephropathy.
Pericyte; myofibroblast; fibrosis; interstitium; diabetic nephropathy