Endothelial nitric oxide synthase controls the expression of the angiogenesis inhibitor thrombospondin 2

Abstract

Injury-and ischemia-induced angiogenesis is critical for tissue repair and requires nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS). We present evidence that NO induces angiogenesis by modulating the level of the angiogenesis inhibitor thrombospondin 2 (TSP2). TSP2 levels were higher than WT in eNOS KO tissues in hind-limb ischemia and cutaneous wounds. In vitro studies confirmed that NO represses TSP2 promoter activity. Moreover, double-eNOS/TSP2 KO mice were generated and found to rescue the phenotype of eNOS KO mice. Studies in mice with knock-in constitutively active or inactive eNOS on the Akt-1 KO background showed that eNOS activity correlates with TSP2 levels. Our observations of NO-mediated regulation of angiogenesis via the suppression of TSP2 expression provide a description of improved eNOS KO phenotype by means other than restoring NO signaling.wound healing | extracellular matrix | Akt | matrix metalloproteinases E ndothelial nitric oxide synthase (eNOS) and its bioactive product nitric oxide (NO) are well-established proangiogenic molecules. Endothelial-derived NO is crucial for maintenance of proper vasodilatory tone and regulation of an antiproliferative and antiapoptotic state for endothelial cells (ECs) and has essential roles in physiological angiogenesis (1-3). Pharmacological inhibition or genetic disruption of eNOS limited angiogenesis during tissue repair, resulting in delayed wound closure (2, 4). Addition of NO donors to wounds enhanced angiogenesis and accelerated healing (5-7). eNOS KO mice recovered poorly from hind-limb ischemia as a consequence of decreased angiogenesis (3,8). These mice also displayed accelerated atherosclerosis, neointimal thickening postinjury, and hypertension (1, 9). Taken together, these observations highlight the ability of eNOS-derived NO to influence vascular function.Thrombospondins (TSPs) are a small family of antiangiogenic matricellular proteins (10). TSPs enhance clearance of matrix metalloproteinase (MMP)-2 and MMP-9 (11-13) and interact with cell-surface receptors, including α v β 3 , very low density lipoprotein receptor (VLDLR), CD36, and CD47, to inhibit angiogenesis (14). Further, ultrastructural studies demonstrated that TSP2 influences ECM assembly (15, 16). TSP2 KO mice displayed improved recovery of blood flow following ischemia (17), altered foreign body response (18,19), and accelerated wound healing (16,20,21). In contrast, TSP1 KO mice displayed delayed healing because of insufficient stimulation of inflammation (13). Consistent with these observations, the expression of TSP1 and TSP2 in tissue repair was associated with the inflammatory and repair phases, respectively (13, 16). Recently, several studies linked components of the Akt-eNOS cascade with TSPs. Specifically, TSP1 has been described to blunt the ability of NO to activate soluble guanyl cyclase (sGC) (22, 23) through interactions with CD36 and CD47 during ischemia. TSP1 has also been described to diminish eNOS activity by blocking phosphorylation at S117...

Up-regulation of Thrombospondin-2 in Akt1-null Mice Contributes to Compromised Tissue Repair Due to Abnormalities in Fibroblast Function

“…These phenotypes were rescued using Akt1 KO mice with a knock-in mutation of eNOS mimicking constitutive activation (S1176D) but not with a loss of function eNOS mutation (S1176A) (49). Interestingly, high TSP2 expression accompanied low eNOS activation associated with the loss of Akt, and the converse was also true (10). In addition, the defective angiogenic phenotype in eNOS KO mice was rescued following the deletion of TSP2 (10).…”

“…TSP2 is not typically expressed at high levels in uninjured tissues, including blood vessels (9,10,18,20), but can be induced after injury, with levels peaking at 10 days of wound healing. TSP2 is undetectable in platelets and endothelial cells, making fibroblasts the primary TSP2 producers in vivo in WT mice (20).…”

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…In contrast to these results in primary vascular and renal cells, treatment of human hepatoma3 B cancer cells with the NO prodrug O2-(2,4-dinitrophenyl) 1-[(4-ethoxyxarbonyl)piperazin-1-yl] diazen-1-ium 1,2-diolate (1 to 10 lM) increased TSP1 mRNA expression and cell surface expression of the TSP1 receptor CD36 (44). Conversely, treatment of NIH 3T3 cells with high concentrations of NO (1 mM DETA/NO) for 24 h suppressed TSP2 promoter activity and mRNA (149).…”

“…Studies in endothelial cells lacking the monooxygenase cytochrome P450 1B1 first implicated regulation of TSP2 expression by eNOSderived NO (266). Genetic evidence for this regulation was later provided using Nos3 -/-mice, which exhibited elevated TSP2 expression in ischemic and wound tissues (149). Studies using Akt1…”

“…The same NOS2 inhibitor prevented downregulation of SMOC1 in kidneys of nephritic rats. Expression of TSP2 in ischemic tissues is regulated by NOS3-dependent NO signaling (149).…”