L-sepiapterin restores SLE serum-induced markers of endothelial function in endothelial cells.

OBJECTIVE: SLE serves as an independent risk factor` for endothelial dysfunction (ED) not explained by Framingham risk factors. We sought to understand the development of SLE-induced ED on a cellular level in order to develop strategies aimed at reversing cellular abnormalities. This study assessed the impact of SLE patient serum on endothelial nitric oxide synthase (eNOS), nitric oxide (NO) production and functional changes in the cell. METHODS: Human umbilical vein endothelial cells (HUVECs) cultured in serum of either SLE (n=25) or healthy patients (n=14) or endothelial basal medium 2 (EBM-2) culture media supplemented with fetal bovine serum with or without L-sepiapterin were used for our studies. We applied the fluorescent probe DAF-FM diacetate for intracellular NO detection using flow cytometry. Total RNA isolates were analysed using reverse transcription PCR for eNOS mRNA expression. Oxygen consumption rate was determined using seahorse analysis. Neutrophil adhesion and migration were determined using a calcein AM microscopy assay. RESULTS: The mRNA expression of eNOS was increased in SLE cultured HUVECs compared with healthy control (p<0.05). The SLE eNOS mRNA level correlated with SLE patient age (p=0.008); however, this trend was not observed with healthy patients. SLE serum reduced NO production in HUVECs compared with EBM-2 cultured cells (p<0.05). Co-treatment of endothelial cells with L-sepiapterin preserved HUVEC capacity to produce NO in SLE conditions (p<0.01). SLE serum enhanced neutrophil migration (p<0.01) but not neutrophil adhesion compared with healthy controls. The bioenergetic health index was not different. CONCLUSIONS: SLE likely causes disruption of endothelial cell eNOS function and NO modulated pathways.

Suppression of angiotensin II-induced pathological changes in heart and kidney by the caveolin-1 scaffolding domain peptide.

Dysregulation of the renin-angiotensin system leads to systemic hypertension and maladaptive fibrosis in various organs. We showed recently that myocardial fibrosis and the loss of cardiac function in mice with transverse aortic constriction (TAC) could be averted by treatment with the caveolin-1 scaffolding domain (CSD) peptide. Here, we used angiotensin II (AngII) infusion (2.1 mg/kg/day for 2 wk) in mice as a second model to confirm and extend our observations on the beneficial effects of CSD on heart and kidney disease. AngII caused cardiac hypertrophy (increased heart weight to body weight ratio (HW/BW) and cardiomyocyte cross-sectional area); fibrosis in heart and kidney (increased levels of collagen I and heat shock protein-47 (HSP47)); and vascular leakage (increased levels of IgG in heart and kidney). Echocardiograms of AngII-infused mice showed increased left ventricular posterior wall thickness (pWTh) and isovolumic relaxation time (IVRT), and decreased ejection fraction (EF), stroke volume (SV), and cardiac output (CO). CSD treatment (i.p. injections, 50 µg/mouse/day) of AngII-infused mice significantly suppressed all of these pathological changes in fibrosis, hypertrophy, vascular leakage, and ventricular function. AngII infusion increased ß1 and ß3 integrin levels and activated Pyk2 in both heart and kidney. These changes were also suppressed by CSD. Finally, bone marrow cell (BMC) isolated from AngII-infused mice showed hyper-migration toward SDF1. When AngII-infused mice were treated with CSD, BMC migration was reduced to the basal level observed in cells from control mice. Importantly, CSD did not affect the AngII-induced increase in blood pressure (BP), indicating that the beneficial effects of CSD were not mediated via normalization of BP. These results strongly indicate that CSD suppresses AngII-induced pathological changes in mice, suggesting that CSD can be developed as a treatment for patients with hypertension and pressure overload-induced heart failure.

Reversal of maladaptive fibrosis and compromised ventricular function in the pressure overloaded heart by a caveolin-1 surrogate peptide.

Chronic ventricular pressure overload (PO) results in congestive heart failure (CHF) in which myocardial fibrosis develops in concert with ventricular dysfunction. Caveolin-1 is important in fibrosis in various tissues due to its decreased expression in fibroblasts and monocytes. The profibrotic effects of low caveolin-1 can be blocked with the caveolin-1 scaffolding domain peptide (CSD, a caveolin-1 surrogate) using both mouse models and human cells. We have studied the beneficial effects of CSD on mice in which PO was induced by trans-aortic constriction (TAC). Beneficial effects observed in TAC mice receiving CSD injections daily included: improved ventricular function (increased ejection fraction, stroke volume, and cardiac output; reduced wall thickness); decreased collagen I, collagen chaperone HSP47, fibronectin, and CTGF levels; decreased activation of non-receptor tyrosine kinases Pyk2 and Src; and decreased activation of eNOS. To determine the source of cells that contribute to fibrosis in CHF, flow cytometric studies were performed that suggested that myofibroblasts in the heart are in large part bone marrow-derived. Two CD45+ cell populations were observed. One (Zone 1) contained CD45+/HSP47-/macrophage marker+ cells (macrophages). The second (Zone 2) contained CD45moderate/HSP47+/macrophage marker- cells often defined as fibrocytes. TAC increased the number of cells in Zones 1 and 2 and the level of HSP47 in Zone 2. These studies are a first step in elucidating the mechanism of action of CSD in heart fibrosis and promoting the development of CSD as a novel treatment to reduce fibrosis and improve ventricular function in CHF patients.