Recent evidence indicates that the SARS-CoV-2 spike protein affects mitochondria with a cell type-dependent outcome. We elucidate the effect of the SARS-CoV-2 receptor binding domain (RBD) on the mitochondrial network and cristae morphology, oxygen consumption, mitoROS production, and inflammatory cytokine expression in cultured human lung microvascular (HLMVECs), coronary artery endothelial (HCAECs), and bronchial epithelial cells (HBECs). Live Mito Orange staining, STED microscopy, and Fiji MiNa analysis were used for mitochondrial cristae and network morphometry; an Agilent XFp analyser for mitochondrial/glycolytic activity; MitoSOX fluorescence for mitochondrial ROS; and qRT-PCR plus Luminex for cytokines. HLMVEC exposure to SARS-CoV-2 RBD resulted in the fragmentation of the mitochondrial network, mitochondrial swelling, increased cristae area, reduced cristae density, and suppressed mitochondrial oxygen consumption and glycolysis. No significant mitochondrial morphology or oxygen consumption changes were observed in HCAECs and HBECs. SARS-CoV-2 RBD induced mitoROS-mediated expression of cytokines GM-CSF and IL-1β in all three investigated cell types, along with IL-8 expression in both endothelial cell types. The findings suggest mitochondrial ROS control SARS-CoV-2 RBD-induced inflammation in HLMVECs, HCAECs, and HBECs, with the mitochondria of HLMVECs being more sensitive to SARS-CoV-2 RBD.

The present study aimed to investigate the effect of a H₂S donor, GYY 4137, on human pulmonary arteries and whether low-frequency ultrasound (20 kHz, 4 W/cm²) inhibits GYY 4137 contractions. Functional studies were conducted on human and rat pulmonary arteries mounted on microvascular myographs. We placed an ultrasonic gadget in the tissue organ bath to insonate the arteries with low-frequency ultrasound. To measure the effect of the low-frequency ultrasound on the entrance of extracellular Ca²⁺, the preparations were placed in a Ca²⁺-free solution, and the thromboxane agonist, U46619, and extracellular calcium were added in the presence of insonation. In isolated human pulmonary arteries, GYY 4137 induced contractions, which were most pronounced in the arteries contracted with the thromboxane analogue, U46619. The transient GYY4137 contractions were reversed by low-frequency ultrasound, a blocker of KV₇ channels, XE-991 (10 µM), and glibenclamide (1 μM), a blocker of ATP-sensitive channels. Low-frequency ultrasound also inhibited the contractions induced by the smooth muscle entrance of increasing extracellular calcium concentrations. The present findings show that GYY 4137 can cause a transient contraction of pulmonary arteries in human arteries. GYY 4137 alone does not cause significant vascular contraction in rat lung arteries, but it contracts rat lung arteries precontracted with U46619. The transient contractions induced by GYY 4137 can be inhibited by low-frequency ultrasound, probably by counteracting the influx of external Ca²⁺. The effect of low-frequency ultrasound counteracts contraction in pulmonary arteries; therefore, a possibility could be to develop a larger device allowing treatment of patients with pulmonary hypertension.

The transient receptor potential vanilloid 4 channel (TRPV4) is thought to play a pivotal role in pulmonary arterial circulation. The present study hypothesizes that TRPV4 activation increases nitric oxide (NO) release and activates calcium-activated potassium of intermediate conductance (KCa3.1) in pulmonary arteries. Pulmonary arteries were isolated from wild-type mice (wt) and mice deficient in KCa3.1 channels (Kcnn4^−/−) and mounted for simultaneous NO concentration and relaxation measurements. Human small pulmonary arteries were isolated and mounted in microvascular myographs for isometric tension recordings. Acetylcholine-induced increases in NO and relaxation of pulmonary arteries were slightly decreased in pulmonary arteries from Kcnn4^−/− versus wt mice. An activator of TRPV4 channels, GSK1016790A, increased NO and relaxation to the same degree in pulmonary arteries from wt and Kcnn4^−/− mice. A blocker of TRPV4 channels, HC06704, inhibited increases in NO concentration with no effect on acetylcholine (ACh) relaxation in pulmonary arteries from wt mice, but blocked increases in NO concentration and relaxation in pulmonary arteries from Kcnn4^−/− mice and responses to GSK1016790A in pulmonary arteries from wt and Kcnn4^−/− mice. Concentration-dependent relaxations induced by an inhibitor of sarcoplasmic Ca-ATPase, cyclopiazonic acid, were blocked in the presence of an inhibitor of NO synthase and a blocker of KCa3.1 channels, TRAM-34, in pulmonary arteries from wt mice, but were unaltered in the presence of TRAM-34 in arteries from Kcnn4^−/− mice, or the presence of a blocker of TRPV4 channels. In small human pulmonary arteries, ACh and sodium nitroprusside (SNP) induced concentration-dependent relaxations, blocked by endothelial cell removal, in the presence of an inhibitor of NO synthase and the KCa3.1 channel blocker TRAM-34. GSK1016790A induced relaxation of human pulmonary arteries with endothelium, but failed to relax arteries without endothelium. The findings suggest that TRPV4 channels are involved in endothelium-dependent relaxation and likely regulate pulmonary vascular tone by modulating NO release.

Endothelial dysfunction is a defining feature of diabetic vascular disease and is characterized by impaired nitric oxide signaling, inflammatory activation, altered mechanotransduction, and disturbed angiogenic responses. The present study investigated whether hyperglycemia modulates endothelial phenotype in a model-dependent manner under distinct structural and mechanical culture conditions. EA.hy926 endothelial cells were cultured for 7 days under normoglycemic (5.5 mM) or high-glucose (25 mM) conditions as static monolayers (G-force = 1 g), adherent clinorotated cells, multicellular spheroids (MCSs) generated during clinorotation, and Matrigel-derived endothelial structures. Gene expression was assessed by qPCR using marker panels related to nitric oxide signaling, PI3K CA-AKT-mTOR signaling, inflammatory adhesion, angiogenesis, and structural adhesion, whereas protein abundance and spatial distribution of eNOS, AKT1, VCAM1, vinculin, and VEGFA together with CDH5/VE-cadherin were analyzed by Western blotting and confocal microscopy. Clinorotation generated both adherent endothelial cells and MCSs. High glucose reduced eNOS-related expression in most models, with the strongest decreases in adherent clinorotated cells and MCSs, whereas Matrigel cultures showed a divergent transcriptional response. PI3K CA expression was markedly suppressed by high glucose in clinostat-derived populations, while mTOR was differentially regulated in spheroids and Matrigel cultures. VCAM1 expression was most prominent in MCSs, whereas ICAM1 was highest in Matrigel cultures. VEGF-related signaling differed substantially among models, and Matrigel cultures showed the strongest VEGFA-associated protein signal and the clearest angiogenic organization under normoglycemic conditions, which became less distinct under high glucose. Vinculin protein abundance was highest in MCSs and Matrigel cultures, reflecting pronounced differences in structural organization. Overall, these findings show that endothelial responses to hyperglycemia are strongly shaped by mechanical and structural context and support the use of complementary in vitro models for studying diabetic endothelial dysfunction.

Background: Endothelial dysfunction is a key feature of diabetic vascular disease and is associated with high-glucose-induced changes in endothelial energy metabolism and redox balance. Transglutaminase-2 has been implicated in vascular stress responses, but its contribution to endothelial adaptation to high glucose remains incompletely defined. Methods: EA.hy926 endothelial cells were cultured under normoglycaemic (NG) or high-glucose conditions and treated with LDN-27219 at 20 µg/mL (48.9 µM). Cellular reducing capacity, total protein content, mitochondrial respiration, MitoSOX Red fluorescence, and selected cytokine and adhesion-marker secretion were assessed. Results: High glucose shifted cells toward a more glycolytic basal phenotype, increased MitoSOX Red fluorescence, and reduced glycolytic stress responsiveness compared with NG conditions. LDN-27219 had limited effects under NG conditions but, under high-glucose conditions, reduced basal respiration, maximal respiration, ATP-linked oxygen consumption, and spare respiratory capacity, while partially restoring stressed extracellular acidification. LDN-27219 also reduced high-glucose-induced MitoSOX Red fluorescence, although this cannot be interpreted as definitive evidence of reduced mitochondrial superoxide production because mitochondrial membrane potential was not measured. No statistically significant changes were detected in the selected inflammatory or adhesion markers. Conclusions: LDN-27219 modifies endothelial bioenergetic and oxidant-associated fluorescence responses under high-glucose conditions.