Elevation of vitreous leptin in diabetic retinopathy and retinal detachment.

PURPOSE: Leptin is a cytokine that regulates energy metabolism and is linked to diabetes mellitus through its metabolic actions. Leptin is angiogenic and promotes wound healing, and therefore this investigation was conducted to determine whether leptin is associated with neovascular and fibrotic complications of diabetes and other retinopathies. METHODS: Serum and vitreous samples were collected from patients classified by the presence and type of diabetic retinopathy or other ocular diseases. Leptin was measured in serum and vitreous by radioimmunoassay, and leptin and leptin receptor were localized in epiretinal membranes immunohistochemically. RESULTS: Leptin levels in serum and vitreous were higher in patients with diabetes than in those without, and vitreous leptin concentrations were especially elevated in patients with proliferative diabetic retinopathy or retinal detachment. Leptin and leptin receptor were detected in fibrovascular epiretinal membrane of patients with diabetes. CONCLUSIONS: Leptin in human vitreous is elevated in proliferative diabetic retinopathy, and retinal detachment and is present in fibrovascular epiretinal tissue. These data suggest an involvement of leptin in retinal disease.

In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse.

We have identified conditions for forming cultured human umbilical vein endothelial cells (HUVEC) into tubes within a three-dimensional gel that on implantation into immunoincompetent mice undergo remodeling into complex microvessels lined by human endothelium. HUVEC suspended in mixed collagen/fibronectin gels organize into cords with early lumena by 24 h and then apoptose. Twenty-hour constructs, s.c. implanted in immunodeficient mice, display HUVEC-lined thin-walled microvessels within the gel 31 days after implantation. Retroviral-mediated overexpression of a caspase-resistant Bcl-2 protein delays HUVEC apoptosis in vitro for over 7 days. Bcl-2-transduced HUVEC produce an increased density of HUVEC-lined perfused microvessels in vivo compared with untransduced or control-transduced HUVEC. Remarkably, Bcl-2- but not control-transduced HUVEC recruit an ingrowth of perivascular smooth-muscle alpha-actin-expressing mouse cells at 31 days, which organize by 60 days into HUVEC-lined multilayered structures resembling true microvessels. This system provides an in vivo model for dissecting mechanisms of microvascular remodeling by using genetically modified endothelium. Incorporation of such human endothelial-lined microvessels into engineered synthetic skin may improve graft viability, especially in recipients with impaired angiogenesis.

Apoptosis-inducing agents cause rapid shedding of tumor necrosis factor receptor 1 (TNFR1). A nonpharmacological explanation for inhibition of TNF-mediated activation.

Several chemical compounds not known to interact with tumor necrosis factor (TNF) signal transducing proteins inhibit TNF-mediated activation of vascular endothelial cells (EC). Four structurally diverse agents, arachidonyl trifluoromethylketone, staurosporine, sodium salicylate, and C6-ceramide, were studied. All four agents caused EC apoptosis at concentrations that inhibited TNF-induced IkappaBalpha degradation. However, evidence of apoptosis was not evident until after several (e.g. 3-12) hours of treatment, whereas 2 h of treatment was sufficient to inhibit TNF responses. IL-1-induced IkappaBalpha degradation was unaffected by these treatments. Inhibition of TNF signaling could not be prevented with either of the broad spectrum caspase inhibitors zVADfmk or yVADcmk. The inhibition of p38 kinase with SB203580 prevented the inhibition of TNF signaling by all agents except arachidonyl trifluoromethylketone. No changes in the levels or molecular weights of the adaptor proteins TRADD (TNF receptor-associated death domain), RIP (receptor-interacting protein), or TRAF2 (TNF receptor-associated factor-2) were caused by apoptogenic drugs. However, TNF receptor 1 (TNFR1) surface expression was significantly reduced by all four agents. Furthermore, TNF-dependent recruitment of TRADD to surface TNFR1 was also inhibited. These data suggest that several putative inhibitors of TNF signaling work by triggering apoptosis and that an early event coincident with the initiation of apoptosis, preceding evidence of injury, is loss of TNFR1. Consistent with this hypothesis, cotreatment of EC with the metalloproteinase inhibitor Tapi (TNF-alpha proteinase inhibitor) blocked the reduction in surface TNFR1 by apoptogenic drugs and prevented inhibition of TNF-induced IkappaBalpha degradation without blocking apoptosis. TNFR1 loss could be a mechanism to limit inflammation in response to apoptotic cell death.

Biological action of leptin as an angiogenic factor.

Leptin is a hormone that regulates food intake, and its receptor (OB-Rb) is expressed primarily in the hypothalamus. Here, it is shown that OB-Rb is also expressed in human vasculature and in primary cultures of human endothelial cells. In vitro and in vivo assays revealed that leptin has angiogenic activity. In vivo, leptin induced neovascularization in corneas from normal rats but not in corneas from fa/fa Zucker rats, which lack functional leptin receptors. These observations indicate that the vascular endothelium is a target for leptin and suggest a physiological mechanism whereby leptin-induced angiogenesis may facilitate increased energy expenditure.

"Cytosolic" phospholipase A2 is in the nucleus of subconfluent endothelial cells but confined to the cytoplasm of confluent endothelial cells and redistributes to the nuclear envelope and cell junctions upon histamine stimulation.

The synthesis of arachidonic acid metabolites is initiated by activation of the sn-arachidonyl-dependent, 85-kd "cytosolic" phospholipase A2 (cPLA2) enzyme. We have investigated the subcellular localization of cPLA2 in resting and histamine-treated human and bovine endothelial cells (EC) using confocal immunofluorescence microscopy. In tightly confluent EC, cPLA2 was primarily localized in the cytoplasm. Surprisingly, in subconfluent EC, cPLA2 was also prominently located within the cell nucleus. By immunoblotting of cell lysates after SDS-PAGE, the cytoplasmic molecular species in subconfluent cells displayed the characteristic Mr 110,000, whereas nuclear extracts contained a predominant Mr 70,000. Nuclear localization of cPLA, in subconfluent EC is independent of cell cycle because it was observed in growth-arrested cells as well as in dividing cells. Nuclear localization was also observed in subconfluent MDCK and HeLa cells where, in contrast to EC, it persisted in tightly confluent monolayers. Treatment of subconfluent EC with histamine caused a rapid, dose-dependent redistribution of cPLA2, from the nucleus to the nuclear envelope. The same treatment of confluent EC produced translocation of cytoplasmic enzyme to the nuclear envelope and to the plasma membrane at the intercellular junctions. The cell density dependence of cPLA2, localization may contribute to altered arachidonic acid metabolism in injured vessels as compared with quiescent vessels.

Suppression of interleukin-1 action by phospholipase-A2 inhibitors in helper T lymphocytes.

The mechanism by which interleukin 1 (IL-1) and T cell receptor (TCR) activation co-stimulate T helper (Th) cells is not clear. In chondrocytes, fibroblasts and several other cell types, much of the evidence suggests a linkage between IL-1 action and increased phospholipase-A2 (PLA2) activity. Although Th cells have very low levels of PLA2, they are well known targets for IL-1. We studied the effects of PLA2 inhibitors, i.e., lipocortin-1 (LC-1) and the synthetic peptide antiflammin-P2 (AF-2), on the enhancing effect of IL-1 upon activation of TCR. When murine D10.G4.1 Th2 cells were stimulated by their clonotypic anti-TCR antibody 3D3, both LC-1 and AF-2 inhibited the biological response to IL-1. This blockade was not seen when D10 cells were induced to proliferate with interleukin-2 (IL-2) and 3D3 in the presence of the same inhibitors, LC-1 or AF-2. These results strongly suggest that PLA2 also plays a central role in mediating the actions of IL-1 in the helper T cell.

T cell receptor-independent immunosuppression induced by dexamethasone in murine T helper cells.

The immune and inflammatory responses are largely inhibited by glucocorticosteroids. In thymocytes, for example, glucocorticosteroids cause apoptosis, whereas they suppress the activity of phospholipase A2 and the production of eicosanoids in tissues actively engaged in inflammation. The immunosuppressive action of dexamethasone (DEX) was studied in vitro by employing a model cell system, namely the murine Th2 clone D10.G4.1 (D10) and its clonotypic anti-T cell receptor (TCR) mAb 3D3. Although the proliferative response of D10 cells to 3D3 stimulation was not affected by DEX, the costimulation provided by IL-1 was dramatically inhibited. Substitution of 3D3 by exogenous IL-4 (as the IL-1 costimulant) failed to prevent the inhibition of proliferation caused by DEX. Yet, when 3D3-mediated stimulation of TCR was supplemented with IL-2, D10 cells were capable of proliferating, even in the presence of DEX. Thus, TCR stimulation on D10 cells remained intact and their resulting propagation was not compromised by DEX treatment. These results provide evidence that immunosuppression caused by DEX is TCR independent and involves an early cytokine-signalling event.