Pericytes protect rats and mice from sepsis-induced injuries by maintaining vascular reactivity and barrier function:implication of miRNAs and microvesicles

Background Vascular hyporeactivity and leakage are key pathophysiologic features that produce multi-organ damage upon sepsis.We hypothesized that pericytes,a group of pluripotent cells that maintain vascular integrity and tension,are protective against sepsis via regulating vascular reactivity and permeability.Methods We conducted a series of in vivo experiments using wild-type(WT),platelet-derived growth factor receptor-β(PDGFR-β)-Cre+mT/mG transgenic mice and Tie2-Cre+Cx43^(flox/flox)mice to examine the relative contribution of pericytes in sepsis,either induced by cecal ligation and puncture(CLP)or lipopolysaccharide(LPS)challenge.In a separate set of experiments with Sprague-Dawley(SD)rats,pericytes were depleted using CP-673451,a selective PDGFR-βinhibitor,at a dosage of 40 mg/(kg·d)for 7 consecutive days.Cultured pericytes,vascular endothelial cells(VECs)and vascular smooth muscle cells(VSMCs)were used for mechanistic investigations.The effects of pericytes and pericyte-derived microvesicles(PCMVs)and candidate miRNAs on vascular reactivity and barrier function were also examined.Results CLP and LPS induced severe injury/loss of pericytes,vascular hyporeactivity and leakage(P<0.05).Transplantation with exogenous pericytes protected vascular reactivity and barrier function via microvessel colonization(P<0.05).Cx43 knockout in either pericytes or VECs reduced pericyte colonization in microvessels(P<0.05).Additionally,PCMVs transferred miR-145 and miR-132 to VSMCs and VECs,respectively,exerting a protective effect on vascular reactivity and barrier function after sepsis(P<0.05).miR-145 primarily improved the contractile response of VSMCs by activating the sphingosine kinase 2(Sphk2)/sphingosine-1-phosphate receptor(S1PR)1/phosphorylation of myosin light chain 20 pathway,whereas miR-132 effectively improved the barrier function of VECs by activating the Sphk2/S1PR2/zonula occludens-1 and vascular endothelial-cadherin pathways.Conclusions Pericytes are protective against sepsis through regulating vascular reactivity and barrier function.Possible mechanisms include both direct colonization of microvasculature and secretion of PCMVs.

Impaired pericyte-Müller glia interaction via PDGFRβ suppression aggravates photoreceptor loss in a rodent model of light-induced retinal injury

AIM:To investigate the involvement of pericyte-Müller glia interaction in retinal damage repair and assess the influence of suppressing the platelet-derived growth factor receptorβ(PDGFRβ)signaling pathway in retinal pericytes on photoreceptor loss and Müller glial response.METHODS:Sprague-Dawley rats were exposed to intense light to induce retinal injury.Neutralizing antibody against PDGFRβwere deployed to block the signaling pathway in retinal pericytes through intravitreal injection.Retinal histology and Müller glial reaction were assessed following light injury.In vitro,normal and PDGFRβ-blocked retinal pericytes were cocultured with Müller cell line(rMC-1)to examine morphological and protein expression changes upon supplementation with light-injured supernatants of homogenized retinas(SHRs).RESULTS:PDGFRβblockage 24h prior to intense light exposure resulted in a significant exacerbation of photoreceptor loss.The upregulation of GFAP and p-STAT3,observed after intense light exposure,was significantly inhibited in the PDGFRβblockage group.Fur ther upregulation of cytokines monocyte chemoattractant protein 1(MCP-1)and interleukin-1β(IL-1β)was also observed following PDGFRβinhibition.In the in vitro coculture system,the addition of light-injured SHRs induced pericyte deformation and upregulation of proliferating cell nuclear antigen(PCNA)expression,while Müller cells exhibited neuron-like morphology and expressed Nestin.However,PDGFRβblockage in retinal pericytes abolished these cellular responses to light-induced damage,consistent with the in vivo PDGFRβblockage findings.CONCLUSION:Pericyte-Müller glia interaction plays a potential role in the endogenous repair process of retinal injury.Impairment of this interaction exacerbates photoreceptor degeneration in light-induced retinal injury.

Biology and function of pericytes in the vascular microcirculation

Pericytes are the main cellular components of tiny arteries and capillaries.Studies have found that pericytes can undergo morphological contraction or relaxation under stimulation by cytokines,thus affecting the contraction and relaxation of microvessels and playing an essential role in regulating vascular microcirculation.Moreover,due to the characteristics of stem cells,pericytes can differentiate into a variety of inflammatory cell phenotypes,which then affect the immune function.Additionally,pericytes can also participate in angiogenesis and wound healing by interacting with endothelial cells in vascular microcirculation disorders.Here we review the origin,biological phenotype and function of pericytes,and discuss the potential mechanisms of pericytes in vascular microcirculation disorders,especially in pulmonary hypertension,so as to provide a sound basis and direction for the prevention and treatment of vascular microcirculation diseases.

TNFSF15 facilitates the differentiation of CD11b^(+) myeloid cells into vascular pericytes in tumors

Objective:Immature vasculature lacking pericyte coverage substantially contributes to tumor growth,drug resistance,and cancer cell dissemination.We previously demonstrated that tumor necrosis factor superfamily 15(TNFSF15)is a cytokine with important roles in modulating hematopoiesis and vascular homeostasis.The main purpose of this study was to explore whether TNFSF15 might promote freshly isolated myeloid cells to differentiate into CD11b^(+) cells and further into pericytes.Methods:A model of Lewis lung cancer was established in mice with red fluorescent bone marrow.After TNFSF15 treatment,CD11b^(+) myeloid cells and vascular pericytes in the tumors,and the co-localization of pericytes and vascular endothelial cells,were assessed.Additionally,CD11b^(+) cells were isolated from wild-type mice and treated with TNFSF15 to determine the effects on the differentiation of these cells.Results:We observed elevated percentages of bone marrow-derived CD11b^(+)myeloid cells and vascular pericytes in TNFSF15-treated tumors,and the latter cells co-localized with vascular endothelial cells.TNFSF15 protected against CD11b^(+)cell apoptosis and facilitated the differentiation of these cells into pericytes by down-regulating Wnt3a-VEGFR1 and up-regulating CD49e-FN signaling pathways.Conclusions:TNFSF15 facilitates the production of CD11b^(+) cells in the bone marrow and promotes the differentiation of these cells into pericytes,which may stabilize the tumor neovasculature.

Wrapping up the role of pericytes in Parkinson's disease

Pericytes are classically defined as contra ctile cells within the central nervous system that regulate blood flow and permeability of the blood-brain barrier(BBB).This one-sided view is gradually changing,and pericytes are now considered versatile cells that can switch their function in response to different stimuli(Uemura et al.,2020).In addition to their role as gatekeepers of the BBB and maintaining homeostasis of the brain’s microenvironment through adj usting the vascular intraluminal dia meter,pericytes are both sensors and initiators of inflammation.

Pericyte-like differentiation of human adipose-derived mesenchymal stem cells:An in vitro study

BACKGROUND Adipose-derived mesenchymal stem cells(ASCs)are characterized by long-term self-renewal and a high proliferation rate.Under adequate conditions,they may differentiate into cells belonging to mesodermal,endodermal or ectodermal lineages.Pericytes support endothelial cells and play an important role in stabilizing the vessel wall at the microcirculation level.The loss of pericytes,as occurs in diabetic retinopathy,results in a breakdown of the blood-retina barrier(BRB)and infiltration of inflammatory cells.In this context,the use of pericytelike differentiated ASCs may represent a valuable therapeutic strategy for restoring BRB damage.AIM To test in vitro strategies to obtain pericyte-like differentiation of human ASCs(hASCs).METHODS Different culture conditions were tested:hASCs cultured in a basal medium supplemented with transforming growth factorβ1;and hASCs cultured in a specific pericyte medium(PM-hASCs).In a further sample,pericyte growth supplement was omitted from the PM.In addition,cultures of human retinal pericytes(hRPCs)were used for comparison.Pericyte-like differentiation of hASCs was tested by immunocytochemical staining and western blotting to evaluate the expression ofα-smooth muscle actin(α-SMA)and neural/glial antigen 2(NG2).Interactions between human retinal endothelial cells(hRECs)and different groups of hASCs were investigated in co-culture experiments.In these cases,the expression of typical junctional proteins such as vascular endothelial-Cadherin,zonula occludens-1 and Occludin were assessed in hRECs.In an in vitro model of the BRB,values of trans-endothelial electrical resistance were measured when hRECs were co-cultured with various groups of pretreated hASCs.The values observed were compared with co-cultures of hRECs and hRPCs as well as with cultures of hRECs alone.Three-dimensional co-cultures of hRECs and hRPCs or pericyte-like hASCs in Matrigel were designed to assess their reciprocal localization.RESULTS After 3-6 d of culture,α-SMA and NG2 immunocytochemistry showed that the closest pericyte-like phenotype was observed when hASCs were cultured in Pericyte Medium(PM-hASCs).In particular,α-SMA immunoreactivity,already visible at the basal level in pericytes and ASCs,was strongly increased only when transforming growth factor was added to the culture medium.NG2 expression,almost undetectable in most conditions,was substantially increased only in PMhASCs.Immunocytochemical results were confirmed by western blot analysis.The presence of pericyte growth supplement seems to increase NG2 expression rather thanα-SMA,in agreement with its role in maintaining pericytes in the proliferative state.In co-culture experiments,immunoreactivity of vascular endothelial-Cadherin,zonula occludens-1 and Occludin was considerably increased in hRECs when hRPCs or PM-hASCs were also present.Supporting results were found by trans-endothelial electrical resistance measurements,gathered at 3 and 6 d of co-culture.The highest resistance values were obtained when hRECs were co-cultured with hRPCs or PM-hASCs.The pericyte-like phenotype of PM-hASCs was also confirmed in three-dimensional co-cultures in Matrigel,where PM-hASCs and hRPCs similarly localized around the tubular formations made by hRECs.CONCLUSION PM-hASCs seem able to strengthen the intercellular junctions between hRECs,likely reinforcing the BRB;thus,hASC-based therapeutic approaches may be developed to restore the integrity of retinal microcirculation.

Exosomes derived from bone marrow mesenchymal stem cells protect the injured spinal cord by inhibiting pericyte pyroptosis

Mesenchymal stem cell(MSC)transplantation is a promising treatment strategy for spinal cord injury,but immunological rejection and possible tumor formation limit its application.The therapeutic effects of MSCs mainly depend on their release of soluble paracrine factors.Exosomes are essential for the secretion of these paracrine effectors.Bone marrow mesenchymal stem cell-derived exosomes(BMSC-EXOs)can be substituted for BMSCs in cell transplantation.However,the underlying mechanisms remain unclear.In this study,a rat model of T10 spinal cord injury was established using the impact method.Then,30 minutes and 1 day after spinal cord injury,the rats were administered 200μL exosomes via the tail vein(200μg/mL;approximately 1×106 BMSCs).Treatment with BMSC-EXOs greatly reduced neuronal cell death,improved myelin arrangement and reduced myelin loss,increased pericyte/endothelial cell coverage on the vascular wall,decreased bloodspinal cord barrier leakage,reduced caspase 1 expression,inhibited interleukin-1βrelease,and accelerated locomotor functional recovery in rats with spinal cord injury.In the cell culture experiment,pericytes were treated with interferon-γand tumor necrosis factor-α.Then,Lipofectamine 3000 was used to deliver lipopolysaccharide into the cells,and the cells were co-incubated with adenosine triphosphate to simulate injury in vitro.Pre-treatment with BMSC-EXOs for 8 hours greatly reduced pericyte pyroptosis and increased pericyte survival rate.These findings suggest that BMSC-EXOs may protect pericytes by inhibiting pyroptosis and by improving blood-spinal cord barrier integrity,thereby promoting the survival of neurons and the extension of nerve fibers,and ultimately improving motor function in rats with spinal cord injury.All protocols were conducted with the approval of the Animal Ethics Committee of Zhengzhou University on March 16,2019.

Versatile subtypes of pericytes and their roles in spinal cord injury repair,bone development and repair

Vascular regeneration is a challenging topic in tissue repair. As one of the important components of the neurovascular unit(NVU),pericytes play an essential role in the maintenance of the vascular network of the spinal cord. To date, subtypes of pericytes have been identified by various markers, namely the PDGFR-β, Desmin, CD146, and NG2, each of which is involved with spinal cord injury(SCI) repair. In addition, pericytes may act as a stem cell source that is important for bone development and regeneration, whilst specific subtypes of pericyte could facilitate bone fracture and defect repair. One of the major challenges of pericyte biology is to determine the specific markers that would clearly distinguish the different subtypes of pericytes, and to develop efficient approaches to isolate and propagate pericytes. In this review, we discuss the biology and roles of pericytes, their markers for identification, and cell differentiation capacity with a focus on the potential application in the treatment of SCI and bone diseases in orthopedics.

Harnessing the stem cell properties of pericytes to repair the brain

Over the last ten years or so,it has become apparent that pericytes have the potential to differentiate into other cell types which may help in the repair and regeneration of tissue after injury.In fact,pericytes have been described as a precursor to mesenchymal stem cells.Their location at the interface between the microvasculature and the brain parenchyma means they are ideally positioned to initiate repair and regeneration in response to various factors.In this perspective,we will highlight how pericytes have stem cell potential alongside their role in regulating processes,such as angiogenesis and inflammation,and discuss how pericytes could be harnessed to promote tissue repair in the brain(Figure 1).

Temporal alterations in pericytes at the acute phase of ischemia/reperfusion in the mouse brain

Pericytes,as the mural cells surrounding the microvasculature,play a critical role in the regulation of microcirculation;however,how these cells respond to ischemic stroke remains unclear.To determine the temporal alterations in pericytes after ischemia/reperfusion,we used the 1-hour middle cerebral artery occlusion model,which was examined at 2,12,and 24 hours after reperfusion.Our results showed that in the reperfused regions,the cerebral blood flow decreased and the infarct volume increased with time.Furthermore,the pericytes in the infarct regions contracted and acted on the vascular endothelial cells within 24 hours after reperfusion.These effects may result in incomplete microcirculation reperfusion and a gradual worsening trend with time in the acute phase.These findings provide strong evidence for explaining the“no-reflow”phenomenon that occurs after recanalization in clinical practice.

Comparison of skeletal and soft tissue pericytes identifies CXCR4+ bone forming mural cells in human tissues

Human osteogenic progenitors are not precisely defined,being primarily studied as heterogeneous multipotent cell populations and termed mesenchymal stem cells(MSCs).Notably,select human pericytes can develop into bone-forming osteoblasts.Here,we sought to define the differentiation potential of CD146 f human pericytes from skeletal and soft tissue sources,with the underlying goal of defining cell surface markers that typify an osteoblastogenic pericyte.CD146+CD31~CD45_pericytes were derived by fluorescence-activated cell sorting from human periosteum,adipose,or dermal tissue.Periosteal CD146+CD31—CD45 cells retained canonical features of pericytes/MSC.Periosteal pericytes demonstrated a striking tendency to undergo osteoblastogenesis in vitro and skeletogenesis in vivo,while soft tissue pericytes did not readily.Transcriptome analysis revealed higher CXCR4 signaling among periosteal pericytes in comparison to their soft tissue counterparts,and CXCR4 chemical inhibition abrogated ectopic ossification by periosteal pericytes.Conversely,enrichment of CXCR4+pericytes or stromal cells identified an osteoblastic/non-adipocytic precursor cell.In sum,human skeletal and soft tissue pericytes differ in their basal abilities to form bone.Diversity exists in soft tissue pericytes,however,and CXCR4+pericytes represent an osteoblastogenic,non-adipocytic cell precursor.Indeed,enrichment for CXCR4-expressing stromal cells is a potential new tactic for skeletal tissue engineering.

Effects of DDPH on HECCM-induced Proliferation and Immunophenotypes of the Pulmonary Vascular Pericytes

In order to study the effects of 1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino) propane hydrochloride (DDPH) on proliferation and immunophenotypes of newborn rat pulmonary vascular pericytes induced by hypoxic endothelial cell conditioned medium (HECCM) from porcine pulmonary arteries, the cultured pericytes were divided into 4 groups according to the endothelial cell conditioned medium (ECCM) used: normoxic ECCM (NECCM) group, NECCM+DDPH group, HECCM group and HECCM+DDPH group. Cell culture, immunocytochemical staining, image analysis and flow cytometric method were used to investigate the effects of HECCM and DDPH on the expression of α-smooth muscle actin (α-SM-Actin) antigen, CD34 antigen, S-100 antigen and proliferating cell nuclear antigen (PCNA) and cell cycle in pericytes. The results showed that the α-SM-Actin antigen in the pericytes in HECCM group was stronger positively expressed than in the other three groups, but CD34 antigen and S-100 antigen were negatively expressed. The expression of α-SM-Actin antigen, CD34 antigen and S-100 antigen was positive in the groups of NECCM, NECCM+DDPH and HECCM+DDPH; The expression of α-SM-Actin and PCNA in HECCM group was 1.32 times (P<0.01) and 1.24 times (P<0.05) that in NECCM group, 1.30 times (P<0.01) and 1.21 times (P<0.05) that in HECCM+DDPH group, respectively. The percentage of the cells in the GO-G1 phase in the HECCM group was lower by 11.7 % and 9.1 %, in S phase higher by 5.6 % and 4.2 %, in G2-M phase higher by 6.1 % and 4.9 % than in the groups of NECCM,HECCM+DDPH, respectively. The inhibitory rate of DDPH on the increased α-SM-Actin and PCNA syntheses in pericytes induced by HECCM were 23.4 % and 17.1 % respectively. The inhibitory rate on the increased pericytes from GO-G1 phase to S phase was 8.3 %. These results suggest that DDPH can directly inhibit pericytes from proliferation and immunophenotypical transformation of smooth muscle-like cells induced by HECCM.

Mechanism of retinal pericyte migration through Angiopoietin/Tie-2 signaling pathway on diabetic rats

AIM： To investigate the mechanism of pericyte migration through Angiopoietin-2 （Ang-2）/Tie-2 signaling pathway. METHODS： We divided the rats into 5 groups. Each diabetic rat model groups injected with Tie-2 inhibitor, ERK1/2 inhibitor, Akt/PKB inhibitor, and DMSO intravitreal. Retinal digest preparation was done to examine the retinal vasculature including pericyte： endothelial ratio, and morphology of pericyte migration. Tie-2, ERKI/2 and Akt/PKB phosporylation were analyzed by confocal laser scanning microscopy. RESULTS： There was a correlation between pericyte migration with increasing Ang-2 （P〈0.05）. Pericyte number reduced by 40% （1：2.4） after 5wk diabetes on diabetic rats. The pericyte： endothelial ratio on group with Tie-2 inhibitor were 1：1.8. The same result shows on group with Akt/PKB inhibition. ERK1/2 inhibitor group shows the best results of pericyte： endothelial ratio （1：1,7）, Inhibition on Tie-2 receptor decreased the phosphorylation activity of Tie-2, ERK1/2 and Akt/PKB pathway. ERK1/2 inhibition also decreasing the phosphorylation of Tie-2 and Akt/PKB. But on Akt/PKB inhibition, the phosphorylation of Tie-2 and ERK1/2 were relative the same. CONCLUSION： Ang-2 has a role for pericyte migration on diabetic rats through Tie-2 receptor, ERKII2 and Akt/PKB pathways. ERK1/2 is a dominant pathway based on the ability to supress another pathway activity and decreasing pericyte migration on diabetic rats.

Multifaceted roles of pericytesinterorgan interactions

Microvascular dysfunction has been implicated in many diseases such as stroke and diabetes.In addition to the microvascular endothelial cell(EC),the pericyte,a perivascular cell that adheres to the abluminal side of the EC may also be important to ensure proper microvascular function.As a prominent perivascular cell,the pericyte has garnered increasing attention for its multiple functional aspects,especially the pericyte of central nervous system(Yemisci et al.,2009;Armulik et al.,2010;Gaceb et al.,2018).

Effect of C-myc Antisense Oligodeoxynucleotides on Hypoxia-induced Proliferation of Pulmonary Vascular Pericytes

To study the effect of c myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin 11 dUTP labeled cDNA, 3H thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c myc antisense ODNs on expression of c myc gene and proliferating cell nuclear antigen (PCNA), and 3H thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c myc and PCNA ( P <0.01), and elevate 3H thymidine incorporation of PC ( P <0.01), but antisense ODNs could significantly inhibit the expression of c myc and PCNA ( P <0.05), and 3H thymidine incorporation of PC ( P <0.01). It was suggested that hypoxia could promote the proliferation of PC by up regulating the expression of c myc gene, but c myc antisense ODNs could inhibit hypoxia induced proliferation of PC by downregulating the expression of c myc gene.

Establishment of the Culture Technique o f Pulmonary Vascular Pericytes and Its Identification in Rats

Summary: In order to study the cellular origin of muscularization in non muscular arterioles of the lung, the pulmonary vascular pericytes culture was established. The terminal lung tissue of the rat was taken out and minced. Then 0.5 % of type Ⅳ collagenase solution was added for digestion and the microvascular segments were obtained by screening. The targeted cells were cultured by “selective conditioned media”. Under phase contrast microscope, the cultured cells were large in size with ragged margin and numerous pseudopodia, which imparted tubule like structure. There was no contact inhibition in growing cells, so multiple layers developed. When they were confluent, there were morphologically no “hillock and dale” growth pattern as in smooth muscle cells or “weave like” pattern as in fibroblasts. The ultrastructure of cultured cells showed numerous digital processes, moderate amount of rough and smooth endoplasmic reticulum, rich Golgi's apparatus, microfilaments, few lysosomes without myofilaments and dense bodies. Immunohistochemical staining revealed that the cultured pericytes had same kind of cellular skeletal protein, α SM actin, like smooth muscle cells. The cultured cells also exhibited positive reaction to CD 34 antigen and S 100 antigen, which were negative in smooth muscle cells and fibroblasts. The cell growth pattern, ultrastructure and immunological phenotype suggested that the cultured cells had characteristics of vascular pericytes. Pericytes are one of the components of microvascular cells, and the establishment of in vitro culture technique of pericytes is of significance for further exploration of the muscularization of non muscular arterioles in lung and the mechanism of structural remodeling of pulmonary vessels.

Role of the Pericytes of Intra－acinar Pulmonary Artery in the Structural Remodeling of Pulmonary Vessels

Whether or not the pericytes exist in the intra-acinar pulmonary arteries and their normal structure and morphological changes during development of the structural remodeling of pulmonary vessels were observed using a pulmonary hypertension model in rats induced by monocrotaline injection.The results showed that the pericytes in the peripheral pulmonary vessels proliferated and transformed into smooth muscle cells during development of pulmonary hypertension,and at the same time,the pericytes could synthesize and secrete extracellular matrix including collagen,suggesting that the pericytes play an important role in the development of pulmonary hypertension and structural remodeling of the pulmonary vessels.

The Effect of Hypoxia on Expression of Basic Fibroblast Growth Factor in Pulmonary Vascular Pericytes

To examine whether hypoxia exerts effect on the expression of basic fibroblast growth ac- tor (bFGF) in pulmonary vascular pericytes (PC), cell culture, in .citu hybridization with probe of digoxigenin-11-dUTP-labled cDNA, immunocytochemistry and image analysis were employed in this study. The results showed that the expression amount of bFGF mRNA and protein in PC of hypoxia (H) group was 1.31 times (P<0. 01) and 1. 17 times (P<0. 01) that of normoxia (N) group re- spectively. It suggests that hypoxia can directly enhance the expression of bFGF mRNA and protein in PC. Increased expression of bFGF may play an important role in the process of PC proliferation and differentiation of PC into smooth muscle-like cells.

AB042.Pericytes on microvessels lead to vascular dysfunction during retinal ischemia

Background:Pericytes are contractile cells that wrap along the walls of capillaries.In the brain,pericytes play a crucial role in the regulation of capillary diameter and vascular blood flow in response to metabolic demand.During ischemia,it has been suggested that pericytes may constrict capillaries,and that pericytes remain constricted after reperfusion thus resulting in impaired blood flow.Methods:Here,we used a mouse model of retinal ischemia based on ligation of the central retinal artery to characterize the role of pericytes on capillary constriction.Ischemia was induced in transgenic mice carrying the NG2 promoter driving red fluorescent protein expression to selectively visualize pericytes(line NG2:DsRed).Changes in retinal capillary diameter at 1 hr after ischemia were measured ex vivo in whole-mounted retinas from ischemic and control eyes(n=4-6/group)using a stereological approach.Vessels and pericytes were three-dimensionally reconstructed using IMARIS(Bitplane).Furthermore,we used a novel and minimally invasive two-photon microscopy approach that allowed live imaging of microvasculature changes in the retina.Results:Our data show a generalized reduction in capillary diameter in ischemic retinas relative to sham-operated controls in all vascular plexus(ischemia:4.7±0.2μm,control:5.2±0.2µm,student’s t-test,P<0.001).Analysis of the number of capillary constrictions at pericyte locations,visualized in NG2:DsRed mice,demonstrated a substantial increase in ischemic retinas relative to the physiological capillary diameter reductions observed in controls(ischemia:1,038±277 constrictions at pericyte locations,control:60±36 constrictions at pericyte locations,student’s t-test,P<0.01).Live imaging using two-photon microscopy confirmed robust capillary constriction at the level of pericytes on retinal capillaries during ischemia(n=6-8/group).Conclusions:Collectively,our data demonstrate that ischemia promotes rapid pericyte constriction on retinal capillaries causing major microvascular dysfunction in this tissue.To identify the molecular mechanisms underlying the pathological response of pericytes during ischemia,we are currently carrying out experiments in mice and zebrafish to modulate signaling pathways involved in calcium dynamics leading to contractility in these cells.

AB011.Live imaging of retinal pericytes:evidence for early calcium uptake,capillary constriction and vascular dysregulation in ocular hypertension glaucoma

Background:Pericytes are contractile cells that wrap along the walls of capillaries.In the brain,pericytes play a crucial role in the regulation of capillary diameter and vascular blood flow in response to metabolic demand.The contribution of pericytes to microvascular deficits in glaucoma is currently unknown.To address this,we used two-photon excitation microscopy for longitudinal monitoring of retinal pericytes and capillaries in a mouse glaucoma model.Methods:Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of albino mice expressing red fluorescent protein selectively in pericytes(NG2-DsRed).Minimally invasive,multiphoton imaging through the sclera of live NG2-DsRed mice was used to visualize pericytes and capillary diameter at one,two and three weeks after glaucoma induction.In vivo fluctuations in pericyte intracellular calcium were monitored with the calcium indicator Fluo-4.Ex vivo stereological analysis of retinal tissue prior to and after injection of microbeads was used to confirm our in vivo findings.Results:Live two-photon imaging of NG2-DsRed retinas demonstrated that ocular hypertension induced progressive accumulation of intracellular calcium in pericytes.Calcium uptake correlated directly with the narrowing of capillaries in the superficial,inner,and outer vascular plexuses(capillary diameter:naïve control=4.7±0.1μm,glaucoma=4.0±0.1μm,n=5-6 mice/group,Student’s t-test P<0.05).Frequency distribution analysis showed a substantial increase in the number of small-diameter capillaries(≤3μm)and a decrease in larger-diameter microvessels(≥5-9μm)at three weeks after induction of ocular hypertension(n=5-6 mice/group,Student’s t-test P<0.05).Conclusions:Our data support two main conclusions.First,two-photon excitation microscopy is an effective strategy to monitor longitudinal changes in retinal pericytes and capillaries in live animals at glaucoma onset and progression.Second,ocular hypertension triggers rapid intracellular calcium increase in retinal pericytes leading to substantial capillary constriction.This study identifies retinal pericytes as important mediators of early microvascular dysfunction in glaucoma.