Relationship of maternal cytomegalovirus-specific antibody responses and viral load to vertical transmission risk following primary maternal infection in a rhesus macaque model.

Cytomegalovirus (CMV) is the most common congenital infection and cause of birth defects worldwide. Primary CMV infection during pregnancy leads to a higher frequency of congenital CMV (cCMV) than maternal re-infection, suggesting that maternal immunity confers partial protection. However, poorly understood immune correlates of protection against placental transmission contributes to the current lack of an approved vaccine to prevent cCMV. In this study, we characterized the kinetics of maternal plasma rhesus CMV (RhCMV) viral load (VL) and RhCMV-specific antibody binding and functional responses in a group of 12 immunocompetent dams with acute, primary RhCMV infection. We defined cCMV transmission as RhCMV detection in amniotic fluid (AF) by qPCR. We then leveraged a large group of past and current primary RhCMV infection studies in late-first/early-second trimester RhCMV-seronegative rhesus macaque dams, including immunocompetent (n = 15), CD4+ T cell-depleted with (n = 6) and without (n = 6) RhCMV-specific polyclonal IgG infusion before infection to evaluate differences between RhCMV AF-positive and AF-negative dams. During the first 3 weeks after infection, the magnitude of RhCMV VL in maternal plasma was higher in AF-positive dams in the combined cohort, while RhCMV glycoprotein B (gB)- and pentamer-specific binding IgG responses were lower magnitude compared to AF-negative dams. However, these observed differences were driven by the CD4+ T cell-depleted dams, as there were no differences in plasma VL or antibody responses between immunocompetent AF-positive vs AF-negative dams. Overall, these results suggest that levels of neither maternal plasma viremia nor humoral responses are associated with cCMV following primary maternal infection in healthy individuals. We speculate that other factors related to innate immunity are more important in this context as antibody responses to acute infection likely develop too late to influence vertical transmission. Yet, pre-existing CMV glycoprotein-specific and neutralizing IgG may provide protection against cCMV following primary maternal CMV infection even in high-risk, immunocompromised settings.

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

Transcriptional signatures of Zika virus infection in astrocytes.

Astrocytes are an early and important target of Zika virus (ZIKV) infection in the developing brain, but the impacts of infection on astrocyte function remain controversial. Given that nonhuman primate (NHP) models of ZIKV infection replicate aspects of neurologic disease seen in human infections, we cultured primary astrocytes from the brain tissue of infant rhesus macaques and then infected the cells with Asian or African lineage ZIKV to identify transcriptional patterns associated with infection in these cells. The African lineage virus appeared to have greater infectivity and promote stronger antiviral signaling, but infection by either strain ultimately produced typical virus response patterns. Both viruses induced hypoxic stress, but the Asian lineage strain additionally had an effect on metabolic and lipid biosynthesis pathways. Together, these findings describe an NHP astrocyte model that may be used to assess transcriptional signatures following ZIKV infection.

Acute Viral Escape Selectively Impairs Nef-Mediated Major Histocompatibility Complex Class I Downmodulation and Increases Susceptibility to Antiviral T Cells.

Nef-specific CD8(+) T lymphocytes (CD8TL) are associated with control of simian immunodeficiency virus (SIV) despite extensive nef variation between and within animals. Deep viral sequencing of the immunodominant Mamu-B*017:01-restricted Nef165-173IW9 epitope revealed highly restricted evolution. A common acute escape variant, T170I, unexpectedly and uniquely degraded Nef's major histocompatibility complex class I (MHC-I) downregulatory capacity, rendering the virus more vulnerable to CD8TL targeting other epitopes. These data aid in a mechanistic understanding of Nef functions and suggest means of immunity-mediated control of lentivirus replication.

Rhesus Cytomegalovirus-encoded Fcγ-binding glycoproteins facilitate viral evasion from IgG-mediated humoral immunity.

Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro , but their role in infection and pathogenesis is unknown. To examine the in vivo function of vFcγRs in animal hosts closely related to humans, we identified and characterized vFcγRs encoded by rhesus CMV (RhCMV). We demonstrate that Rh05, Rh152/151 and Rh173 represent the complete set of RhCMV vFcγRs, each displaying functional similarities to their respective HCMV orthologs with respect to antagonizing host FcγR activation in vitro . When RhCMV-naïve rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma viremia levels and anti-RhCMV antibody responses were comparable to wildtype infections. However, the duration of plasma viremia was significantly shortened in immunocompetent, but not in CD4+ T cell-depleted animals. Since vFcγRs were not required for superinfection, we conclude that vFcγRs delay control by virus-specific adaptive immune responses, particularly antibodies, during primary infection.

The pentameric complex is not required for vertical transmission of cytomegalovirus in seronegative pregnant rhesus macaques.

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neonatal neurological impairment but essential virological determinants of transplacental CMV transmission remain unclear. The pentameric complex (PC), composed of five subunits, glycoproteins H (gH), gL, UL128, UL130, and UL131A, is essential for efficient entry into non-fibroblast cells in vitro . Based on this role in cell tropism, the PC is considered a possible target for CMV vaccines and immunotherapies to prevent cCMV. To determine the role of the PC in transplacental CMV transmission in a non-human primate model of cCMV, we constructed a PC-deficient rhesus CMV (RhCMV) by deleting the homologues of the HCMV PC subunits UL128 and UL130 and compared congenital transmission to PC-intact RhCMV in CD4+ T cell-depleted or immunocompetent RhCMV-seronegative, pregnant rhesus macaques (RM). Surprisingly, we found that the transplacental transmission rate was similar for PC-intact and PC-deleted RhCMV based on viral genomic DNA detection in amniotic fluid. Moreover, PC-deleted and PC-intact RhCMV acute infection led to similar peak maternal plasma viremia. However, there was less viral shedding in maternal urine and saliva and less viral dissemination in fetal tissues in the PC-deleted group. As expected, dams inoculated with PC-deleted RhCMV demonstrated lower plasma IgG binding to PC-intact RhCMV virions and soluble PC, as well as reduced neutralization of PC-dependent entry of the PC-intact RhCMV isolate UCD52 into epithelial cells. In contrast, binding to gH expressed on the cell surface and neutralization of entry into fibroblasts by the PC-intact RhCMV was higher for dams infected with PC-deleted RhCMV compared to those infected with PC-intact RhCMV. Our data demonstrates that the PC is dispensable for transplacental CMV infection in our non-human primate model. One Sentence Summary: Congenital CMV transmission frequency in seronegative rhesus macaques is not affected by the deletion of the viral pentameric complex.

Relationship of maternal cytomegalovirus-specific antibody responses and viral load to vertical transmission risk following primary maternal infection in a rhesus macaque model.

Cytomegalovirus (CMV) is the most common congenital infection and cause of birth defects worldwide. Primary CMV infection during pregnancy leads to a higher frequency of congenital CMV (cCMV) than maternal re-infection, suggesting that maternal immunity confers partial protection. However, poorly understood immune correlates of protection against placental transmission contributes to the current lack of an approved vaccine to prevent cCMV. In this study, we characterized the kinetics of maternal plasma rhesus CMV (RhCMV) viral load (VL) and RhCMV-specific antibody binding and functional responses in a group of 12 immunocompetent dams with acute, primary RhCMV infection. We defined cCMV transmission as RhCMV detection in amniotic fluid (AF) by qPCR. We then leveraged a large group of past and current primary RhCMV infection studies in late-first/early-second trimester RhCMV-seronegative rhesus macaque dams, including immunocompetent (n=15), CD4+ T cell-depleted with (n=6) and without (n=6) RhCMV-specific polyclonal IgG infusion before infection to evaluate differences between RhCMV AF-positive and AF-negative dams. During the first 3 weeks after infection, the magnitude of RhCMV VL in maternal plasma was higher in AF-positive dams in the combined cohort, while RhCMV glycoprotein B (gB)- and pentamer-specific binding IgG responses were lower magnitude compared to AF-negative dams. However, these observed differences were driven by the CD4+ T cell-depleted dams, as there were no differences in plasma VL or antibody responses between immunocompetent AF-positive vs AF-negative dams. Overall, these results suggest that levels of neither maternal plasma viremia nor humoral responses are associated with cCMV following primary maternal infection in healthy individuals. We speculate that other factors related to innate immunity are more important in this context as antibody responses to acute infection likely develop too late to influence vertical transmission. Yet, pre-existing CMV glycoprotein-specific and neutralizing IgG may provide protection against cCMV following primary maternal CMV infection even in high-risk, immunocompromised settings.

Immunomodulatory potential of in vivo natural killer T (NKT) activation by NKTT320 in Mauritian-origin cynomolgus macaques.

Invariant natural killer T-lymphocytes (iNKT) are unique immunomodulatory innate T cells with an invariant TCRα recognizing glycolipids presented on MHC class-I-like CD1d molecules. Activated iNKT rapidly secrete pro-and anti-inflammatory cytokines, potentiate immunity, and modulate inflammation. Here, we report the effects of in vivo iNKT activation in Mauritian-origin cynomolgus macaques by a humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple plasma analytes within 24 hours. Flow cytometry and RNA-Seq confirmed downstream activation of multiple immune subsets, enrichment of JAK/STAT and PI3K/AKT pathway genes, and upregulation of inflammation-modulating genes. NKTT320 also increased iNKT frequency in adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, which supports its future use as an immunotherapeutic.

CYP1B1 expression promotes the proangiogenic phenotype of endothelium through decreased intracellular oxidative stress and thrombospondin-2 expression.

Reactive species derived from cell oxygenation processes play an important role in vascular homeostasis and the pathogenesis of many diseases including retinopathy of prematurity. We show that CYP1B1-deficient (CYP1B1(-/-)) mice fail to elicit a neovascular response during oxygen-induced ischemic retinopathy. In addition, the retinal endothelial cells (ECs) prepared from CYP1B1(-/-) mice are less adherent, less migratory, and fail to undergo capillary morphogenesis. These aberrant cellular responses were completely reversed when oxygen levels were lowered or an antioxidant added. CYP1B1(-/-) ECs exhibited increased oxidative stress and expressed increased amounts of the antiangiogenic factor thrombospondin-2 (TSP2). Increased lipid peroxidation and TSP2 were both observed in retinas from CYP1B1(-/-) mice and were reversed by administration of an antioxidant. Reexpression of CYP1B1 in CYP1B1(-/-) ECs resulted in down-regulation of TSP2 expression and restoration of capillary morphogenesis. A TSP2 knockdown in CYP1B1(-/-) ECs also restored capillary morphogenesis. Thus, CYP1B1 metabolizes cell products that modulate intracellular oxidative stress, which enhances production of TSP2, an inhibitor of EC migration and capillary morphogenesis. Evidence is presented that similar changes occur in retinal endothelium in vivo to limit neovascularization.

Immune Profile of the Normal Maternal-Fetal Interface in Rhesus Macaques and Its Alteration Following Zika Virus Infection.

The maternal decidua is an immunologically complex environment that balances maintenance of immune tolerance to fetal paternal antigens with protection of the fetus against vertical transmission of maternal pathogens. To better understand host immune determinants of congenital infection at the maternal-fetal tissue interface, we performed a comparative analysis of innate and adaptive immune cell subsets in the peripheral blood and decidua of healthy rhesus macaque pregnancies across all trimesters of gestation and determined changes after Zika virus (ZIKV) infection. Using one 28-color and one 18-color polychromatic flow cytometry panel we simultaneously analyzed the frequency, phenotype, activation status and trafficking properties of αß T, γδ T, iNKT, regulatory T (Treg), NK cells, B lymphocytes, monocytes, macrophages, and dendritic cells (DC). Decidual leukocytes showed a striking enrichment of activated effector memory and tissue-resident memory CD4+ and CD8+ T lymphocytes, CD4+ Tregs, CD56+ NK cells, CD14+CD16+ monocytes, CD206+ tissue-resident macrophages, and a paucity of B lymphocytes when compared to peripheral blood. t-distributed stochastic neighbor embedding (tSNE) revealed unique populations of decidual NK, T, DC and monocyte/macrophage subsets. Principal component analysis showed distinct spatial localization of decidual and circulating leukocytes contributed by NK and CD8+ T lymphocytes, and separation of decidua based on gestational age contributed by memory CD4+ and CD8+ T lymphocytes. Decidua from 10 ZIKV-infected dams obtained 16-56 days post infection at third (n=9) or second (n=1) trimester showed a significant reduction in frequency of activated, CXCR3+, and/or Granzyme B+ memory CD4+ and CD8+ T lymphocytes and γδ T compared to normal decidua. These data suggest that ZIKV induces local immunosuppression with reduced immune recruitment and impaired cytotoxicity. Our study adds to the immune characterization of the maternal-fetal interface in a translational nonhuman primate model of congenital infection and provides novel insight in to putative mechanisms of vertical transmission.

PECAM-1 regulates proangiogenic properties of endothelial cells through modulation of cell-cell and cell-matrix interactions.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a member of the immunoglobulin superfamily of cell adhesion molecules with important roles in angiogenesis and inflammation. However, the molecular and cellular mechanisms, and the role that specific PECAM-1 isoforms play in these processes, remain elusive. We recently showed attenuation of retinal vascular development and neovascularization in PECAM-1-deficient (PECAM-1-/-) mice. To gain further insight into the role of PECAM-1 in these processes, we isolated primary retinal endothelial cells (EC) from wild-type (PECAM-1+/+) and PECAM-1-/- mice. Lack of PECAM-1 had a significant impact on endothelial cell-cell and cell-matrix interactions, resulting in attenuation of cell migration and capillary morphogenesis. Mechanistically these changes were associated with a significant decrease in expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) bioavailability in PECAM-1-/- retinal EC. PECAM-1-/- retinal EC also exhibited a lower rate of apoptosis under basal and challenged conditions, consistent with their increased growth rate. Furthermore, reexpression of PECAM-1 was sufficient to restore migration and capillary morphogenesis of null cells in an isoform-specific manner. Thus PECAM-1 expression modulates proangiogenic properties of EC, and these activities are significantly influenced by alternative splicing of its cytoplasmic domain.

CYP1B1 and endothelial nitric oxide synthase combine to sustain proangiogenic functions of endothelial cells under hyperoxic stress.

We have recently shown that deletion of constitutively expressed CYP1B1 is associated with attenuation of retinal endothelial cell (EC) capillary morphogenesis (CM) in vitro and angiogenesis in vivo. This was largely caused by increased intracellular oxidative stress and increased production of thrombospondin-2, an endogenous inhibitor of angiogenesis. Here, we demonstrate that endothelium nitric oxide synthase (eNOS) expression is dramatically decreased in the ECs prepared from retina, lung, heart, and aorta of CYP1B1-deficient (CYP1B1(-/-)) mice compared with wild-type (CYP1B1(+/+)) mice. The eNOS expression was also decreased in retinal vasculature of CYP1B1(-/-) mice. Inhibition of eNOS activity in cultured CYP1B1(+/+) retinal ECs blocked CM and was concomitant with increased oxidative stress, like in CYP1B1(-/-) retinal ECs. In addition, expression of eNOS in CYP1B1(-/-) retinal ECs or their incubation with a nitric oxide (NO) donor enhanced NO levels, lowered oxidative stress, and improved cell migration and CM. Inhibition of CYP1B1 activity in the CYP1B1(+/+) retinal ECs resulted in reduced NO levels and attenuation of CM. In contrast, expression of CYP1B1 increased NO levels and enhanced CM of CYP1B1(-/-) retinal ECs. Furthermore, attenuation of CYP1B1 expression with small interfering RNA proportionally lowered eNOS expression and NO levels in wild-type cells. Together, our results link CYP1B1 metabolism in retinal ECs with sustained eNOS activity and NO synthesis and/or bioavailability and low oxidative stress and thrombospondin-2 expression. Thus CYP1B1 and eNOS cooperate in different ways to lower oxidative stress and thereby to promote CM in vitro and angiogenesis in vivo.

Opposing effects of bim and bcl-2 on lung endothelial cell migration.

Integration of cell adhesive, survival, and proliferative processes is essential for capillary morphogenesis of endothelial cells (EC) in vitro and vascular development and function in vivo. Unfortunately, the molecular and cellular mechanisms that impact these processes are poorly defined. Here we examined how lack of bim and/or bcl-2 expression impact lung EC function. The absence of bcl-2 or bim had a significant impact on EC adhesion and migration. Lack of bcl-2 expression decreased lung EC migration, whereas lack of bim expression increased migration compared with their wild-type counterparts. Decreased adhesion to fibronectin and vitronectin was observed in both bcl-2-/- and bim-/- lung EC, with bcl-2-/- EC having very little adhesion to either matrix protein. Capillary morphogenesis was greatly diminished in bcl-2-/- EC, which correlated with decreased lung alveolarization in vivo, an angiogenesis-dependent process. We also observed aberrant production of extracellular matrix proteins, eNOS expression, and nitric oxide production in bcl-2-/- lung EC, which could contribute to inability to undergo capillary morphogenesis. The changes in cell adhesion and migration noted in the absence of bim or bcl-2 were independent of their impact on apoptosis. We observed no significant affect on the steady-state rate of apoptosis of lung EC in the absence of bim or bcl-2. Thus, bcl-2 family members, bim and bcl-2, play a central role in modulation of EC proangiogenic properties, which goes beyond their role as simple mediators of mitochondrial homeostasis and apoptosis.

Immune outcomes of Zika virus infection in nonhuman primates.

Although the Zika virus (ZIKV) epidemic is subsiding, immune responses that are important for controlling acute infection have not been definitively characterized. Nonhuman primate (NHP) models were rapidly developed to understand the disease and to test vaccines, and these models have since provided an understanding of the immune responses that correlate with protection during natural infection and vaccination. Here, we infected a small group of male rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques with a minimally passaged Brazilian ZIKV isolate and used multicolor flow cytometry and transcriptional profiling to describe early immune patterns following infection. We found evidence of strong innate antiviral responses together with induction of neutralizing antibodies and T cell responses. We also assessed the relative importance of CD8 T cells in controlling infection by carrying out CD8 T cell depletion in an additional two animals of each species. CD8 depletion appeared to dysregulate early antiviral responses and possibly increase viral persistence, but the absence of CD8 T cells ultimately did not impair control of the virus. Together, these data describe immunological trends in two NHP species during acute ZIKV infection, providing an account of early responses that may be important in controlling infection.

Attenuation of retinal vascular development and neovascularization in PECAM-1-deficient mice.

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) is expressed on the surface of endothelial cells (EC) at high levels with important roles in angiogenesis and inflammation. However, the physiological role PECAM-1 plays during vascular development and angiogenesis remains largely unknown. Here we determined the role of PECAM-1 in the postnatal development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) using PECAM-1-deficient (PECAM-1-/-) mice. A significant decrease in retinal vascular density was observed in PECAM-1-/- mice compared with PECAM-1+/+ mice. This was attributed to a decreased number of EC in the retinas of PECAM-1-/- mice. An increase in the rate of apoptosis was observed in retinal vessels of PECAM-1-/- mice, which was compensated, in part, by an increase in the rate of proliferation. However, the development and regression of hyaloid vasculature were not affected in the absence of PECAM-1. We did not observe a significant defect in astrocytes, the number of endothelial tip cell filopodias, and the rate of developing retinal vasculature progression in PECAM-1-/- mice. However, we observed aberrant organization of arterioles and venules, decreased secondary branching, and dilated vessels in retinal vasculature of PECAM-1-/- mice. In addition, retinal neovascularization was attenuated in PECAM-1-/- mice during OIR despite an expression of VEGF similar to that of PECAM-1+/+ mice. Mechanistically, these changes were associated with an increase in EphB4 and ephrin B2, and a decrease in eNOS, expression in retinal vasculature of PECAM-1-/- mice. These results suggest that PECAM-1 expression and its potential interactions with EphB4/ephrin B2 and eNOS are important for survival, migration, and functional organization of EC during retinal vascular development and angiogenesis.

A fluorescence polarization assay for identifying ligands that bind to vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a homodimeric proangiogenic protein that induces endothelial cell migration and proliferation primarily through interactions with its major receptors, VEGFR-1 and VEGFR-2. Inhibitors of one or both of these VEGF-receptor interactions could be beneficial as therapeutics for diseases caused by dysfunctional angiogenesis (e.g., cancer). Others have reported small peptides that bind to the VEGF dimer at surface regions that are recognized by the receptors. Here we report the development of a fluorescence polarization assay based on the binding to VEGF of a derivative of one of these peptides that has been labeled with BODIPY-tetramethylrhodamine (BODIPY(TMR)). This 384-well format assay is tolerant to dimethyl sulfoxide (DMSO, up to 4% [v/v]) and has a Z' factor of 0.76, making it useful for identifying molecules that associate with the receptor-binding surface of the VEGF dimer.

Calcitriol is a potent inhibitor of retinal neovascularization.

PURPOSE: Vitamin D compounds inhibit the growth of a variety of tumors in preclinical and clinical studies. Among the mechanisms suggested for this inhibition is antiangiogenesis. Retinal angiogenesis is the basis for vision loss in several major blinding diseases. The purpose of this study was to evaluate the antiangiogenic activity of calcitriol (1,25-dihydroxyvitamin D(3)) in vivo and its effect on retinal endothelial cell (EC) proliferation, migration, and capillary morphogenesis in vitro. METHODS: The mouse oxygen-induced ischemic retinopathy (OIR) model was used to assess the antiangiogenic activity of calcitriol. Ocular VEGF levels were determined by Western blot analysis of whole eye extracts from postnatal day (P) 15 mice during OIR. The effects of calcitriol on retinal EC proliferation, migration, and capillary morphogenesis were also assessed in vitro. RESULTS: Calcitriol-treated animals demonstrated a significant decrease in retinal neovascularization compared with control animals. This effect was dose dependent, and retinal neovascularization was significantly inhibited in calcitriol-treated mice. Although no deaths occurred, calcitriol administration was associated with increased serum calcium and a lack of increase in body weight in a dose-independent manner. The ocular level of VEGF was similar in control and calcitriol-treated animals. At a lower concentration of calcitriol, retinal EC capillary morphogenesis in solubilized basement membrane was inhibited without a significant inhibitory effect on EC proliferation and migration. The concentration of calcitriol required to inhibit retinal EC proliferation was significantly higher than that required to inhibit EC capillary morphogenesis. CONCLUSIONS: These data suggest calcitriol is a potent inhibitor of retinal neovascularization and may be of benefit in the treatment of a variety of eye diseases with a neovascular component.

Isolation and characterization of corneal endothelial cells from wild type and thrombospondin-1 deficient mice.

PURPOSE: To isolate and characterize primary corneal endothelial cells (CEC) from wild type and transgenic mice to facilitate the study of their properties in vitro. METHODS: CEC were isolated from wild type or transgenic-immortomice corneas. The Descemet's membrane was gently peeled from the periphery of the cornea towards the central region and placed into wells of a 96 well tissue culture plate coated with fibronectin in growth medium. Cells that grew out were trypsinized and expanded on fibronectin-coated wells and used for further characterization. CEC were evaluated for expression and localization of specific markers and adhesion molecules by FACS analysis and indirect immunofluorescence staining. The migration properties of CEC were evaluated using a scratch wound and transwell assay, while their ability to undergo capillary morphogenesis was assessed on Matrigel. RESULTS: Isolation of CEC from transgenic mice has been somewhat challenging and not previously reported. Here we describe a method for isolation of CEC from wild type and thrombospondin-1 deficient (TSP1-/-) immortomice. Our results indicate that nearly 100% of selected cells express B4-lectin and VE-cadherin, but not PECAM-1. These cells were successfully passaged and maintained in culture for several months without a significant loss in expression of these markers. The wild type CEC, like vascular EC, organized and formed a capillary-like cell network on Matrigel. The ability of the CEC from TSP1-/- mice to form such a network was somewhat compromised. This may be attributed, at least in part, to altered adhesive and migratory properties of these cells. CONCLUSIONS: The CEC can be readily obtained from wild type and transgenic mice, which facilitate the comparison and identification of the physiologic role of specific genes in CEC function.

Isolation and characterization of murine retinal astrocytes.

PURPOSE: To isolate and characterize primary retinal astrocytes in culture (RAC) from wild-type and transgenic mice to aid the study of their properties in vitro. METHODS: Astrocytes were isolated from wild-type and transgenic Immortomice by collagenase digestion of the retina. Affinity purification using magnetic beads coated with anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) was used to remove retinal endothelial cells. The remaining cells were cultured and expanded. The majority of these cells were identified as astrocytes. These cells were characterized for expression of astrocytic markers using fluorescence-activated cell sorting (FACS) and immunostaining analysis. The expression of various integrins and other cell adhesion molecules on the surface of retinal astrocytes, their adhesion to various matrix proteins, their migration, and their ability to organize on Matrigel were determined. RESULTS: Here we describe a method for the isolation of RAC from wild-type and thrombospondin-1 deficient (TSP1-/-) mice. Our results indicated that nearly 100% of cells isolated expressed the astrocytic markers GFAP, NG2, Pax2, and vimentin. These cells were successfully passaged and maintained in culture for several months without a significant loss in expression of astrocytic markers. The RAC expressed alphavbeta3 integrin and other cell adhesion molecules on their surface. The TSP1-/- RAC adhered more strongly to fibronectin and vitronectin compared to the wild-type cells, while neither cell types adhered to collagen and laminin. Wild-type and TSP1-/- RAC exhibited similar migratory characteristics despite alterations in their adhesive properties and production of various matrix proteins. Also, these cells, like endothelial cells, similarly organized into a network in Matrigel. CONCLUSIONS: The RAC can be readily obtained from wild-type and transgenic mice. This facilitates the comparison and identification of specific gene functions in RAC compared to astrocytes prepared from other sites of central nervous system.

Attenuation of proliferation and migration of retinal pericytes in the absence of thrombospondin-1.

Perivascular supporting cells, including vascular smooth muscle cells (VSMCs) and pericytes (PCs), provide instructive signals to adjacent endothelial cells helping to maintain vascular homeostasis. These signals are provided through direct contact and by the release of soluble factors by these cells. Thrombospondin (TSP)1 is a matricellular protein and an autocrine factor for VSMCs. TSP1 activity, along with that of PDGF, regulates VSMC proliferation and migration. However, the manner in which TSP1 and PDGF impact retinal PC function requires further investigation. In the present study, we describe, for the first time, the isolation and culture of retinal PCs from wild-type (TSP1(+/+)) and TSP1-deficient (TSP1(-/-)) immortomice. We showed that these cells express early and mature markers of PCs, including NG2, PDGF receptor-beta, and smooth muscle actin as well as desmin, calbindin, and mesenchymal stem cell markers. These cells were successfully passaged and maintained in culture for several months without significant loss of expression of these markers. TSP1(+/+) PCs proliferated at a faster rate compared with TSP1(-/-) PCs. In addition, TSP1(+/+) PCs, like VSMCs, responded to PDGF-BB with enhanced migration and proliferation. In contrast, TSP1(-/-) PCs failed to respond to the promigratory and proliferative activity of PDGF-BB. This may be attributed, at least in part, to the limited interaction of PDGF-BB with TSP1 in null cells, which is essential for PDGF proliferative and migratory action. We observed no significant differences in the rates of apoptosis in these cells. TSP1(-/-) PCs were also less adherent, expressed increased levels of TSP2 and fibronectin, and had decreased amounts of N-cadherin and alpha(v)beta(3)-integrin on their surface. Thus, TSP1 plays a significant role in retinal PC proliferation and migration impacting retinal vascular development and homeostasis.