Direct activation of endothelial cells by SARS-CoV-2 nucleocapsid protein is blocked by Simvastatin

Emerging evidence suggests that endothelial activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multi-organ failure in patients with COVID-19. However, the molecular mechanisms underlying endothelial activation in COVID-19 patients remain unclear. In this study, the SARS-CoV-2 viral proteins that potently activate human endothelial cells were screened to elucidate the molecular mechanisms involved with endothelial activation. It was found that nucleocapsid protein (NP) of SARS-CoV-2 significantly activated human endothelial cells through TLR2/NF-{kappa}B and MAPK signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Remarkablely, though the protein sequences of N proteins from coronaviruses are highly conserved, only NP from SARS-CoV-2 induced endothelial activation. The NPs from other coronaviruses such as SARS-CoV, MERS-CoV, HUB1-CoV and influenza virus H1N1 did not affect endothelial activation. These findings are well consistent with the results from clinical investigations showing broad endotheliitis and organ injury in severe COVID-19 patients. In conclusion, the study provides insights on SARS-CoV-2-induced vasculopathy and coagulopathy, and suggests that simvastatin, an FDA-approved lipid-lowering drug, may benefit to prevent the pathogenesis and improve the outcome of COVID-19 patients.

The role of calcineurin in the lung fibroblasts proliferation and collagen synthesis induced by basic fibroblast growth factor / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the role of calcineurin (CaN) in the lung fibroblast proliferation and collagen synthesis induced by basic fibroblast growth factor (bFGF).</p><p><b>METHODS</b>We used Western blot and immunohistochemical methods for investigating the content and distribution of calcineurin in the lung tissue. Calcineurin activity in different tissues was measured using (32)P-labelled substrate. In the primary culture of lung fibroblasts, (3)H-thymidine ((3)H-TdR) and (3)H-proline incorporation methods were used to study the effect of cyclosporin A (CsA), an inhibitor of calcineurin, on the lung fibroblast DNA and collagen synthesis stimulated by bFGF.</p><p><b>RESULTS</b>We found that calcineurin was expressed in lung tissue and has phosphatase activity (7.1 +/- 2.0 pmol Pi/mg pr/min). CsA (10(-8) - 10(-6) mol/L) inhibited lung fibroblast (3)H-TdR incorporation induced by bFGF in a dose-dependent manner, with the inhibitory rates by 20%, 46% and 66% (P < 0.01). CsA (10(-7) - 10(-6) mol/L) inhibited (3)H-proline incorporation in lung fibroblasts stimulated by bFGF, with the inhibitory rates by 21% and 37% (P < 0.01). In a culture medium, CsA (10(-8) - 10(-6) mol/L) inhibited (3)H-proline secretion induced by bFGF in a dose-dependent manner, with the inhibitory rates by 19%, 29% (P < 0.05) and 56% (P < 0.01). CsA (10(-7) mol/L) could inhibit calcineurin activity by 44% in lung fibroblasts (P < 0.01).</p><p><b>CONCLUSIONS</b>Calcineurin is expressed in lung tissue and has phosphatase activity. It is involved in the bFGF stimulated lung fibroblast DNA and collagen synthesis.</p>

Alteration of functional receptors in heart for human urotensin II in pressure overload- induced cardiac hypertrophy of rats / 中国病理生理杂志

AIM: To investigate alteration of functional receptors for urotensin II in pressure overload-induced cardiac hypertrophy of rats. METHODS: In the rat cardiac hypertrophy model produced by constriction of the abdominal aorta, urotensin II binding sites in myocardial sarcolemma were determined with radioligand assay first day (early group) and 30th day (late group) after operation. RESULTS: Compared with control and sham group, in the early group，the blood pressure increased 54% and 43% respectively(P＜0.01),and heart/body weight ratio unchanged(P＞0.05),UII receptors were up-regulated by 184% and 159%(P＜0.01)respectively, While the affinity to UII was decreased (Kd increased 224% and 206 respectively,P＜0.01).In the late group, the blood pressure increased 85% and 67% (P＜0.01), and heart/body weight ratio increased 18% and 22% (P＜0.05) respectively，than that of control and sham group. UII receptors were down-regulated by 35% and 41%(P＜0.05) respectively while the affinity to UII was increased (Kd decreased 30% and 33% respectively, P＜0.05). CONCLUSION: The biphasic changes of UII receptor in myocardial sarcolemma induced by pressure-overload were observed,increasing in early stage, while decreasing in late stage, and these changes were involved in the development of cardiac hypertrophy.

Distribution and activity of calcineurin in different tissues of rat / 中国病理生理杂志

AIM: To investigate the activity and distribution of calcineurin (CaN) in different tissues of rat. METHODS: Using western blot and immunohistochemical staining methods to measure the amount and location of CnA?, isoform of catalytic subunit of CaN in different tissues of rat. CaN activity was measured by labelled substrate peptide. RESULTS: 1. Western blot showed that CnA? expression was detected in brain, heart, skeletal muscle and lung tissues. There was no detectable CnA? expression in kidney and aorta. 2. In immunohistochemical staining study, there was strong immunostaining of CnA? in brain. CnA? was located in cytoplasm of cardiac cell, macrophage and connective tissue of peribronchiolar in lung tissue, aorta adventitia, connective tissue around small vessels and outer wall of renal tube. 3. CaN activity was highest in brain, the following was skeletal muscle, myocardium and lung tissue. CaN activity was lowest in aorta and kidney tissue. CONCLUSION: CaN is widely distributed in rats and might be involved in functional regulation of various organs and tissues.

Metallothionein involvement in myocardial protection of basic fibroblast growth factor / 中国病理生理杂志

AIM: To observe whether metallothionein plays a role in cardiac protective effect of basic fibroblast growth factor on anoxic/reperfusion (A/R) injury in cultured cardiomyocytes and study the possible mechanism of cardiac protection by bFGF.METHODS: The present study made the model of myocyte A/R injury after having a 24 h incubation by bFGF(10 -10、10 -9、10 -8 mol/L) and bFGF(10 -9 mol/L)+PD 098059 respectively. We measured the levels of MT and MDA in myocytes, and the changes of LDH and protein in cultured medium. We also counted the number of viable cell in groups.RESULTS: The contents of myocardial MT were significantly increased after treatment by bFGF. The levels of MT in 10 -10 mol/L、10 -9 mol/L and 10 -8 mol/L bFGF treated groups increased 54%\, 62%\, 76% respectively, compared with the A/R group, and the number of viable cell were also greatly increased, LDH and protein leakage in cultured medium and MDA contents in myocyte were dramatically decreased in bFGF treated groups. All the protection were completely disappeared with the inhibition of MT production with PD 098059, the inhibitor of mitogen-activated protein kinase(MAPK).CONCLUSION: MT involves in the protection of bFGF in cultured cardiomyocytes. It might be related with activation of MAPKase.

Role and regulation of calcineurin in angiotensin Ⅱ-stimulated cardiac myocyte hypertrophy of rats / 中国病理生理杂志

AIM: To study the role and regulation of calcineurin(CaN) in angiotensin II(AngⅡ)-stimulated cardiacmyocyte hypertrophy of rats. METHODS: Using AngⅡ to induce the cultured cardiac myocyte hypertrophy of rats, and investigating the effect of CaN inhibitor on [ 3H]-leucine incorporation of AngⅡ-stimulated cardiomyocytes and the regulation of various factors on CaN activity in cardiomyocytes.RESULTS: AngⅡ can stimulate the CaN activity in cultured neonatal rat cardiomyocytes in a dose- and time-dependent manner. In cardiac myocytes incubated with 10, 100, 1000 nmol?L -1 of AngⅡ for 12h, the CaN activities increased respectively by 13%,57%( P

Metallothionein involvement in myocardial protection of basic fibroblast growth factor / 中国病理生理杂志

AIM: To observe whether metallothionein plays a role in cardiac protective effect of basic fibroblast growth factor on anoxic/reperfusion (A/R) injury in cultured cardiomyocytes and study the possible mechanism of cardiac protection by bFGF.METHODS: The present study made the model of myocyte A/R injury after having a 24 h incubation by bFGF( 10-10、10-9、10-s mol/L) and bFGF( 10-9 mol/L) + PD098059 respectively. We measured the levels of MT and MDA in myocytes, and the changes of LDH and protein in cultured medium. We also counted the number of viable cell in groups. RESULTS: The contents of myocardial MT were significantly increased after treatment by bFGF. The levels of MT in I0-l0 mol/L、10-9 mol/L and 10-8 mol/L bFGF treated groups increased 54 %、 62%、 76% respectively, compared with the A/R group, and the number of viable cell were also greatly increased, LDH and protein leakage in cultured medium and MDA contents in myocyte were dramatically decreased in bFGF treated groups. All the protection were completely disappeared with the inhibition of MT production with PD098059, theinhibitor of mitogen- activated protein kinase(MAPK). CONCLUSION: MT involves in the protection of bFGF in cultured cardiomyocytes. It might be related with activation of MAPKase.

Effect of batroxobin on thromboxane level in caninewith myocardial ischemia/reperfusion injury / 中国病理生理杂志

AIM:To investigate the effect of batroxobin on thromboxane A 2(TXA 2)level in canine heart ischemia/reperfusion (I/R) injury and the cardioprotective mechanism of batroxobin against I/R injury. METHODS: Canine heart ischemia was induced by ligation of the left anterior descending coronary artery for 30 min followed by 90 min reperfusion.Batroxobin was intravenously administered before heart ischemia and 15 min before reperfusion.The level of plasma thromboxane (TXA 2) ,creatine phosphokinase (CK), lactic dehydrogenase (LDH ) and the concentration of myocardial TXA 2 were measured. The pathological changes in I/R myocardium were observed. RESULTS: In I/R group, TXA 2 levels of both plasma and myocardium increased significantly,and myocardium was injured obviously. Myocyte cells of central zone of I/R heart showed intracellular edema, swollen and damaged mitochondria, and fragmentation of cristae and concentrated nucleus. Plasma CK and LDH level was also elevated. Both ways of batroxobin administration reduced TXA 2 level apparently and plasma CK?LDH were also reduced significantly. LVEDP lowered while +dp/dt max elevated greatly,the mortality of I/R canine reduced obviously. CONCLUSION: Batroxobin decreased TXA 2 levels of both plasma and myocardium significantly, and could afford protection from I/R injury.

Myocardial fibroblasts of rat are an important source of producing adrenomedullin / 中国病理生理杂志

AIM: To elucidate the cellular source of adrenomedullin (AM) in myocardial tissue, the effects of 5 substances on secretion of AM from cultured rat fibroblasts and myocytes were examined in this study. METHODS: Myocardial fibroblast and myocyte isolation and culture; AM was measured by radioimmunoassay; AM receptors were assayed by radioactive analysis method. RESULTS:Tumor necrosis factor ? (TNF?), lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) all could stimulate AM synthesis and secretion in myocardial Fb and myocyte , and the amount of AM secreted by Fb was greater than that by myocytes, whereas transforming growth factor-?(TGF?),and interferon-?(IFN?) suppressed in it in myocardial Fb and myocytes. Myocardial Fb has two kinds of AM binding sites, but myocyte has only one high affinity- binding site.CONCLUSIOIN:Myocardial fibroblasts could synthesize and secrete AM, and there are AM receptors in both myocardial fibroblasts and myocytes.