Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin (H4B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (O 2 •؊ ). H4B was found recyclable from its oxidized form by dihydrofolate reductase (DHFR) in several cell types. Functionality of the endothelial DHFR, however, remains completely unknown. Here we present findings that specific inhibition of endothelial DHFR by RNA interference markedly reduced endothelial H4B and nitric oxide (NO • ) bioavailability. Furthermore, angiotensin II (100 nmol͞liter for 24 h) caused a H4B deficiency that was mediated by H2O2-dependent downregulation of DHFR. This response was associated with a significant increase in endothelial O 2•؊ production, which was abolished by eNOS inhibitor N-nitro-L-arginine-methyl ester or H2O2 scavenger polyethylene glycol-conjugated catalase, strongly suggesting H2O2-dependent eNOS uncoupling. Rapid and transient activation of endothelial NAD(P)H oxidases was responsible for the initial burst production of O 2 • (Rac1 inhibitor NSC 23766 but not an N-nitro-L-arginine-methyl ester-attenuated ESR O 2•؊ signal at 30 min) in response to angiotensin II, preceding a second peak in O 2•؊ production at 24 h that predominantly depended on uncoupled eNOS. Overexpression of DHFR restored NO • production and diminished eNOS production of O 2•؊ in angiotensin II-stimulated cells. In conclusion, these data represent evidence that DHFR is critical for H4B and NO • bioavailability in the endothelium. Endothelial NAD(P)H oxidase-derived H2O2 down-regulates DHFR expression in response to angiotensin II, resulting in H4B deficiency and uncoupling of eNOS. This signaling cascade may represent a universal mechanism underlying eNOS dysfunction under pathophysiological conditions associated with oxidant stress.hydrogen peroxide ͉ tetrahydrobiopterin ͉ superoxide

It has been shown that eNOS uncoupling occurs in hypertension and atherosclerosis. However its causal role in vascular pathogenesis has not been previously characterized. Here, we challenged eNOS pre-uncoupled hph-1 mice (deficient in eNOS cofactor tetrahydrobiopterin biosynthetic enzyme GTPCHI) with Ang II (0.7 mg/kg/day, 14 days). Both wild-type (WT) and hph-1 groups developed hypertension similarly up to day 6 to 7. Thereafter approximately 14% of Ang II-infused (0.7 mg/kg/day) hph-1 mice (n=72) started to die suddenly of ruptured AAA. Among the survivors, 65% developed AAA, resulting in a total morbidity rate of 79%. In contrast, none of the Ang II-infused WT mice died or developed AAA. Ang II progressively deteriorated eNOS uncoupling in hph-1 mice, while augmenting H4B and nitric oxide (NO•) deficiencies. The abundance of the H4B salvage enzyme dihydrofolate reductase (DHFR) in the endothelium was decreased in hph-1 mice and further diminished by Ang II infusion. Intriguingly, restoration of DHFR expression by oral administration of folic acid (FA), or overexpression of DHFR, completely prevented AAA formation in Ang II-infused hph-1 mice while attenuating progressive uncoupling of eNOS. Folic acid also attenuated vascular remodelling and inflammation characterized by medial elastin break down, augmented MMP2 activity and activation of MMP9, as well as macrophage infiltration. In conclusion, these data innovatively suggest a causal role of eNOS uncoupling/H4B deficiency in AAA formation. Therefore oral FA administration, endothelium targeted DHFR gene therapy, and perhaps other countermeasures directed against eNOS uncoupling, could be used as new therapeutics for AAA.

Background-Folate supplementation improves endothelial function in patients with hyperhomocysteinemia. Mechanistic insights into potential benefits of folate on vascular function in general population however, remain mysterious.

Objective-Microparticles are membrane vesicles with procoagulant and proinflammatory properties released during cell activation, including apoptosis. The present study was designed in dissecting the effects evoked by microparticles on vascular reactivity. Methods and Results-Microparticles from either apoptotic T lymphocytic cells or from plasma of diabetic patients with vascular complications induced vascular hyporeactivity in response to vasoconstrictor agents in mouse aorta. Hyporeactivity was reversed by nitric oxide (NO) synthase plus cyclooxygenase-2 inhibitors, and associated with an increased production of vasodilatory products such as NO and prostacyclin. Microparticles induced an upregulation of proinflammatory protein expressions, inducible NO-synthase and cyclooxygenase-2, mainly in the medial layer of the vessels as evidenced by immunochemical staining. In addition, microparticles evoke NF-B activation probably through the interaction with the Fas/Fas Ligand pathway. Finally, in vivo treatment of mice with lymphocyte-derived MPs induces vascular hyporeactivity, which was reversed by the combination of NO and cyclooxygenase-2 inhibitors. Conclusion-These data provide a rationale to explain the paracrine role of microparticles as vectors of transcellular exchange of message in promoting vascular dysfunction during inflammatory diseases. (Arterioscler Thromb Vasc Biol. 2005;25:2522-2527.)Key Words: Fas/Fas Ligand Ⅲ microvesicles Ⅲ proinflammatory proteins Ⅲ vascular dysfunction Ⅲ vasoactivity R ecent data suggest that inflammation play a central role in the origin and complications of cardiovascular disease. For instance, inflammation has a pivotal role in the development of atherosclerosis and the acute activation of the vascular wall with consecutive local thrombosis and altered vasoactivity. This process is orchestrated by the interactions between inflammatory cells, such as leukocytes and vascular cells, endothelial cells, and smooth muscle cells, which under activation or apoptosis, for example, lead to the release of circulating microparticles (MPs). MPs are vesicles shed from the blebbing plasma membrane of various cell types, such as platelets, T and B cells, monocytes, and endothelial cells during activation by agonists, shear stress, 1,2 or apoptosis. 3,4 MPs harbor cell surface proteins and contain cytoplasmic components of the original cell. They exhibit negatively charged phospholipids, chiefly phosphatidylserine, at their surface accounting for their procoagulant character 1 and proinflammatory properties. 5 Elevated levels of circulating MPs have been detected under pathological states, such as atherosclerosis, acute coronary syndrome, diabetes, preeclampsia, and sepsis. 4,6 -9 Furthermore, these pathologies are associated with vascular dysfunction including attenuation of endothelium-dependent vasodilatation, alteration of responsiveness of vascular smooth muscle to vasoconstrictor stimuli, or both. However, the mechanisms triggering the modifications of the vessel contraction/relaxati...

Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the in vivo roles of the epidermal proteases in NS using a set of mouse models individually or simultaneously deficient for KLK5 and KLK7 on the genetic background of a novel NS-mouse model. We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy.

Background:The gut microbiome and metabolome may significantly influence clinical outcomes in patients with short bowel syndrome (SBS). The study aimed to describe specific metagenomic/metabolomics profiles of different SBS types and to identify possible therapeutic targets. Methods: Fecal microbiome (FM), volatile organic compounds (VOCs), and bile acid (BA) spectrum were analyzed in parenteral nutrition (PN)-dependent SBS I, SBS II, and PN-independent (non-PN) SBS patients. Results: FM in SBS I, SBS II, and non-PN SBS shared characteristic features (depletion of beneficial anaerobes, high abundance of Lactobacilaceae and Enterobacteriaceae). SBS I patients were characterized by the abundance of oxygen-tolerant microrganisms and depletion of strict anaerobes. Non-PN SBS subjects showed markers of partial FM normalization. FM dysbiosis was translated into VOC and BA profiles characteristic for each SBS cohort. A typical signature of all SBS patients comprised high saturated aldehydes and medium-chain fatty acids and reduced short-chain fatty acid (SCFA) content. Particularly, SBS I and II exhibited low protein metabolism intermediate (indole, p-cresol) content despite the hypothetical presence of relevant metabolism pathways. Distinctive non-PN SBS marker was high phenol content. SBS patients' BA fecal spectrum was enriched by chenodeoxycholic and deoxycholic acids and depleted of lithocholic acid. Conclusions: Environmental conditions in SBS gut significantly affect FM composition and metabolic activity. The common feature of diverse SBS subjects is the altered VOC/BA profile and the lack of important products of microbial metabolism. Strategies oriented on the microbiome/metabolome reconstitution and targeted delivery of key compounds may represent a promising therapeutic strategy in SBS patients. (JPEN J Parenter Enteral Nutr. 2020;44:105-118) Keywords bile acids; gut microbiota; parenteral nutrition; short bowel syndrome, short-chain fatty acids, volatile organic compounds From the

FA might serve as a novel therapeutic option combating PH.

Background and purpose: Red wine polyphenols (RWPs) inhibit the expression of vascular endothelial growth factor (VEGF), a major pro-angiogenic and pro-atherosclerotic factor, in vascular smooth muscle cells (VSMCs). The aim of this study was to identify which red wine polyphenols were inhibitory and to determine the mechanism underlying the inhibitory effects. Experimental approach: Release of VEGF stimulated by platelet derived growth factor AB (PDGF AB ) , from human aortic VSMCs was measured by immunoassay and phosphorylation of kinases by Western blot analysis. The direct antioxidant properties of polyphenols were determined by electron paramagnetic resonance and the cellular formation of reactive oxygen species (ROS) by dichlorofluorescein. Key results: The inhibitory effect of RWPs on PDGF AB -induced release of VEGF was mimicked by delphinidin but not by quercetin, catechins, resveratrol, gallic acid or caffeic acid. In the anthocyanin class, not only delphinidin but also cyanidin prevented VEGF release whereas malvidin and peonidin were without effect. RWPs, delphinidin and cyanidin directly scavenged ROS and prevented the PDGF AB -induced formation of ROS in VSMCs. Malvidin and peonidin did not scavenge ROS but prevented the cellular formation of ROS. Although the p38 MAPK, ERK1/2 and JNK pathways have been involved in the PDGF AB -induced expression of VEGF, in our experiments, only phosphorylation of p38 MAPK and JNK was inhibited by RWPs, delphinidin and cyanidin. Conclusions and implications: Anthocyanins presenting a hydroxyl residue at position 3 0 are able to inhibit PDGF AB -induced VEGF expression by preventing activation of p38 MAPK and JNK in VSMCs.