In order to investigate the role of nitrative stress in vascular endothelial injury in hyperhomocysteinemia (HHcy), thirty healthy adult female Wistar rats were randomly divided into three groups: control, hyperhomocysteinemia model, and hyperhomocysteinemia with FeTMPyP (peroxynitrite scavenger) treatment. The endothelium-dependent dilatation of thoracic aorta in vitro was determined by response to acetylcholine (ACh). The histological changes in endothelium were assessed by HE staining and scanning electron microscopy (SEM). The expression of 3-nitrotyrosine (NT) in thoracic aorta was demonstrated by immunohistochemistry and immunofluorescence, and the number of circulating endothelial progenitor cells (EPCs) was quantified by flow cytometry. Hyperhomocysteinemia caused significant endothelial injury and dysfunction including vasodilative and histologic changes, associated with higher expression of NT in thoracic aorta. FeTMPyP treatment reversed these injuries significantly. Further, the effect of nitrative stress on cultured EPCs in vitro was investigated by administering peroxynitrite donor (3-morpholino-sydnonimine, SIN-1) and peroxynitrite scavenger (FeTMPyP). The roles of nitrative stress on cell viability, necrosis and apoptosis were evaluated with 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. Also, the phospho-eNOS expression and tube formation in Matrigel of cultured EPCs was detected. Our data showed that the survival of EPCs was much lower in SIN-1 group than in vehicle group, both the apoptosis and necrosis of EPCs were much more severe, and the p-eNOS expression and tube formation in Matrigel were obviously declined. Subsequent pretreatment with FeTMPyP reversed these changes. Further, pretreatment with FeTMPyP reversed homocysteine-induced EPC injury. In conclusion, this study indicates that nitrative stress plays a role in vascular endothelial injury in hyperhomocysteinemia, as well as induces endothelial progenitor cell injury directly.

The kidneys are important organs that are susceptible to aging. Hyperhomocysteinemia (HHcy) is a risk factor for nephropathy and is associated with chronic nephritis, purpuric nephritis, and nephrotic syndrome. Numerous studies have shown that elevated serum homocysteine levels can damage the kidneys; however, the underlying mechanism of HHcy on kidney damage remains unclear. In this study, we make use of a diet-induced HHcy rat model and in vitro cell culture to explore the role of autophagy in HHcy-induced renal aging and further explored the underlying mechanism. We demonstrated that HHcy led to the development of renal aging. Promoted kidney aging and autophagic insufficiency were involved in HHcy-induced renal aging. HHcy decreased the expression of transcription factor EB (TFEB), the key transcription factor of autophagy-related genes in renal tissue. Further experiments showed that nitrative stress levels were increased in the kidney of HHcy rats. Interestingly, pretreatment with the peroxynitrite (ONOO-) scavenger FeTMPyP not only reduced the Hcy-induced nitrative stress in vitro but also partially attenuated the decrease in TFEB in both protein and mRNA levels. Moreover, our results indicated that HHcy reduced TFEB expression and inhibited TFEB-mediated autophagy activation by elevating nitrative stress. In conclusion, this study showed an important role of autophagic insufficiency in HHcy-induced renal aging, in which downregulation of TFEB plays a major role. Furthermore, downexpression of TFEB was associated with increased nitrative stress in HHcy. This study provides a novel insight into the mechanism and therapeutic strategy for renal aging.

Sp1-CSE-H2S pathway plays an important role in homocysteine-metabolism, whose disorder can result in hyperhomocysteinemia. H2S deficiency in hyperhomocysteinemia has been reported, while the underlying mechanism and whether it in turn affects the progress of hyperhomocysteinemia are unclear. This study focused on the post-translational modification of Sp1/CSE and revealed four major findings: (1) Homocysteine-accumulation augmented CSE's nitration, inhibited its bio-activity, thus caused H2S deficiency. (2) H2S deficiency inhibited the S-sulfhydration of Sp1, down-regulated CSE and decreased H2S further, which in turn weakened CSE's own S-sulfhydration. (3) CSE was S-sulfhydrated at Cys84, Cys109, Cys172, Cys229, Cys252, Cys307 and Cys310, among which the S-sulfhydration of Cys172 and Cys310 didn't affect its enzymatic activity, while the S-sulfhydration of Cys84, Cys109, Cys229, Cys252 and Cys307 was necessary for its bio-activity. (4) H2S deficiency trapped homocysteine-metabolism into a vicious cycle, which could be broken by either blocking nitration or restoring S-sulfhydration. This study detected a new mechanism that caused severe hyperhomocysteinemia, thereby provided new therapeutic strategies for hyperhomocysteinemia.

Hyperhomocysteinemia is strongly associated with cardiovascular diseases. Previous studies have shown that phytoestrogen α-zearalanol can protect cardiovascular system from hyperhomocysteinemia and ameliorate the level of plasma total homocysteine; however, the underlying mechanisms remain to be clarified. The aim of this research is to investigate the possible molecular mechanisms involved in ameliorating the level of plasma homocysteine by α-zearalanol. By the successfully established diet-induced hyperhomocysteinemia rat models, we found that, after α-zearalanol treatment, the activity of cystathionine β-synthase, the key enzyme in homocysteine metabolism, was significantly elevated and level of nitrative stress in liver was significantly reduced. In correlation with this, results also showed a decreased nitration level of cystathionine β-synthase in liver. Together data implied that alleviation of plasma homocysteine level by phytoestrogen α-zearalanol might be related to the reduction of cystathionine β-synthase nitration.

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. The enhanced nitrative stress plays an important role in homocysteine-induced endothelial dysfunction. Previous studies have showed that phytoestrogen α -zearalanol alleviated endothelial injury in ovariectomized hyperhomocysteinemic rats; however, the underlying mechanism remains to be clarified. This study was to investigate the effects of α -zearalanol on homocysteine-induced endothelial apoptosis in vitro and explore the possible role of nitrative stress in these effects. Results showed that homocysteine (500  μ mol/L, 24 h) induced the apoptosis of human umbilical vein endothelial cells (HUVECs) obviously, and this effect was significantly attenuated by pretreatment with α -zearalanol (10(-8)~10(-6) mol/L). Moreover, α -zearalanol downregulated proapoptotic protein Bax, upregulated antiapoptotic proteins Bcl-2 and Bcl-XL, and decreased the expression and activity of caspase-9. These findings demonstrated that α -zearalanol could effectively alleviate homocysteine-induced endothelial apoptosis, and this antiapoptosis effect might be related to the inhibition of the intrinsic pathway. Western blot indicated an enhanced 3-nitrotyrosine expression in HUVECs when challenged with homocysteine, which was attenuated by pretreatment with α -zearalanol. This result implied that inhibition of nitrative stress might play a role in the protective effect of α -zearalanol on endothelial cells. Such discovery may shed a novel light on the antiatherogenic activities of α -zearalanol in hyperhomocysteinemia.

Hyperhomocysteinemia (HHcy) is a well-known independent risk factor for vascular diseases in the general population. This study was to explore the effect of genistein (GST), a natural bioactive compound derived from legumes, on HHcy-induced vascular endothelial impairment in ovariectomized rats in vivo. Thirty-two adult female Wistar rats were assigned randomly into four groups (n = 8): (a) Con: control; (b) Met: 2.5% methionine diet; (c) OVX + Met: ovariectomy + 2.5% methionine diet; (d) OVX + Met + GST: ovariectomy + 2.5% methionine diet + supplementation with genistein. After 12 wk of different treatment, the rats' blood, toracic aortas and liver samples were collected for analysis. Results showed that high-methionine diet induced both elevation of plasma Hcy and endothelial dysfunction, and ovariectomy deteriorated these injuries. Significant improvement of both functional and morphological changes of vascular endothelium was observed in OVX + Met + GST group; meanwhile the plasma Hcy levels decreased remarkably. There were significant elevations of plasma ET-1 and liver MDA levels in ovariectomized HHcy rats, and supplementation with genistein could attenuate these changes. These results implied that genistein could lower the elevated Hcy levels, and prevent the development of endothelial impairment in ovariectomized HHcy rats. This finding may shed a novel light on the anti-atherogenic activities of genistein in HHcy patients.