TNF/iNOS/NO pathway mediates host susceptibility to endothelial-dependent circulatory failure and death induced by betacoronavirus infection.

Poor disease outcomes and lethality are directly related to endothelial dysfunction in betacoronavirus infections. Here, we investigated the mechanisms underlying the vascular dysfunction caused by the betacoronaviruses MHV-3 and SARS-CoV-2. Wild-type C57BL/6 (WT) and knockout mice for inducible nitric oxide synthase (iNOS-/-) or TNF receptor 1 (TNFR1-/-) were infected with MHV-3, and K18-hACE2 transgenic mice expressing human ACE2 were infected with SARS-CoV-2. Isometric tension was used to evaluate vascular function. Protein expression was determined by immunofluorescence. Tail-cuff plethysmography and Doppler were used to assess blood pressure and flow, respectively. Nitric oxide (NO) was quantified with the DAF probe. ELISA was used to assess cytokine production. Survival curves were estimated using Kaplan-Meier. MHV-3 infection reduced aortic and vena cava contractility, arterial blood pressure, and blood flow, resulting in death. Resistance mesenteric arteries showed increased contractility. The contractility of the aorta was normalized by removing the endothelium, inhibiting iNOS, genetically deleting iNOS, or scavenging NO. In the aorta, iNOS and phospho-NF-kB p65 subunit expression was enhanced, along with basal NO production. TNF production was increased in plasma and vascular tissue. Genetic deletion of TNFR1 prevented vascular changes triggered by MHV-3, and death. Basal NO production and iNOS expression were also increased by SARS-CoV-2. In conclusion, betacoronavirus induces an endothelium-dependent decrease in contractility in macro-arteries and veins, leading to circulatory failure and death via TNF/iNOS/NO. These data highlight the key role of the vascular endothelium and TNF in the pathogenesis and lethality of coronaviruses.

Sex differences in the participation of endothelial mediators and signaling pathways involved in the vasodilator effect of a selective GPER agonist in resistance arteries of gonadectomized Wistar rats.

AIM: Endothelial mechanisms underlying the vascular effects of estrogen modulated by the G protein-coupled estrogen receptor (GPER) are not well understood, especially in gonadal sex hormone deprivation. Thus, we investigated vascular function and endothelial signaling pathways involved in the selective activation of GPER in resistance arteries of gonadectomized rats. METHODS: Gonadectomy was performed in Wistar rats of both sexes. After 21 days, the animals were euthanized. Concentration-response curves were obtained by cumulative additions of G-1 in third-order mesenteric arteries. The vasodilatory effects of G-1 were evaluated before and after endothelium removal or incubation with pharmacological inhibitors. Tissue protein expression was measured by western blotting. Assays with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) and 2',7' dichlorodihydrofluorescein-diacetate (H2DCF-DA) were performed in the arteries investigated. Immunolocalization was assessed by immunofluorescence. RESULTS: G-1 induced partially endothelium-dependent relaxation in both sexes. The three isoforms of the enzyme nitric oxide synthase contributed to the production and release of nitric oxide in both gonadectomized groups, but the role of inducible nitric oxide synthase is more expressive in males. The mechanistic pathway by which endothelial nitric oxide synthase is phosphorylated appears to differ between sexes, with the rapid signaling pathway phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3k-Akt-eNOS) being identified for males and mitogen-activated protein kinase/extracellular signal-regulated kinase/endothelial nitric oxide synthase (MEK-ERK-eNOS) for females. The contribution of hydrogen peroxide as an endothelial relaxation mediator seems to be greater in females. CONCLUSION: These results provide new insights into the effects of estrogen-induced responses via GPER on vascular function in gonadal sex hormone deprivation.

Sex Differences in the Vasodilation Mediated by G Protein-Coupled Estrogen Receptor (GPER) in Hypertensive Rats.

BACKGROUND: The protective effect of estrogen on the vasculature cannot be explained only by its action through the receptors ERα and ERß. G protein-coupled estrogen receptors (GPER)-which are widely distributed throughout the cardiovascular system-may also be involved in this response. However, little is known about GPER actions in hypertension. Therefore, in this study we evaluated the vascular response mediated by GPER using a specific agonist, G-1, in spontaneously hypertensive rats (SHR). We hypothesized that G-1 would induce a relaxing response in resistance mesenteric arteries from SHR of both sexes. METHODS: G-1 concentration-response curves (1 nM-10 µM) were performed in mesenteric arteries from SHR of both sexes (10-12-weeks-old, weighing 180-250 g). The effects of G-1 were evaluated before and after endothelial removal and incubation for 30 min with the inhibitors L-NAME (300 µM) and indomethacin (10 µM) alone or combined with clotrimazole (0.75 µM) or catalase (1,000 units/mL). GPER immunolocalization was also investigated, and vascular hydrogen peroxide (H2O2) and ROS were evaluated using dichlorofluorescein (DCF) and dihydroethidium (DHE) staining, respectively. RESULTS: GPER activation promoted a similar relaxing response in resistance mesenteric arteries of female and male hypertensive rats, but with the participation of different endothelial mediators. Males appear to be more dependent on the NO pathway, followed by the H2O2 pathway, and females on the endothelium and H2O2 pathway. CONCLUSION: These findings show that the GPER agonist G-1 can induce a relaxing response in mesenteric arteries from hypertensive rats of both sexes in a similar way, albeit with differential participation of endothelial mediators. These results contribute to the understanding of GPER activation on resistance mesenteric arteries in essential hypertension.

Role of the α7 Nicotinic Acetylcholine Receptor in the Pathophysiology of Atherosclerosis.

Atherosclerosis constitutes a major risk factor for cardiovascular diseases, the leading cause of morbidity and mortality worldwide. This slowly progressing, chronic inflammatory disorder of large- and medium-sized arteries involves complex recruitment of immune cells, lipid accumulation, and vascular structural remodeling. The α7 nicotinic acetylcholine receptor (α7nAChR) is expressed in several cell types involved in the genesis and progression of atherosclerosis, including macrophages, dendritic cells, T and B cells, vascular endothelial and smooth muscle cells (VSMCs). Recently, the α7nAChR has been described as an essential regulator of inflammation as this receptor mediates the inhibition of cytokine synthesis through the cholinergic anti-inflammatory pathway, a mechanism involved in the attenuation of atherosclerotic disease. Aside from the neuronal cholinergic control of inflammation, the non-neuronal cholinergic system similarly regulates the immune function. Acetylcholine released from T cells acts in an autocrine/paracrine fashion at the α7nAChR of various immune cells to modulate immune function. This mechanism additionally has potential implications in reducing atherosclerotic plaque formation. In contrast, the activation of α7nAChR is linked to the induction of angiogenesis and VSMC proliferation, which may contribute to the progression of atherosclerosis. Therefore, both atheroprotective and pro-atherogenic roles are attributed to the stimulation of α7nAChRs, and their role in the genesis and progression of atheromatous plaque is still under debate. This minireview highlights the current knowledge on the involvement of the α7nAChR in the pathophysiology of atherosclerosis.