Vascular mechanisms of post-COVID-19 conditions: Rho-kinase is a novel target for therapy

Abstract Background In post-coronavirus disease-19 (post-COVID-19) conditions (long COVID), systemic vascular dysfunction is implicated, but the mechanisms are uncertain, and the treatment is imprecise. Methods and results Patients convalescing after hospitalization for COVID-19 and risk factor matched controls underwent multisystem phenotyping using blood biomarkers, cardiorenal and pulmonary imaging, and gluteal subcutaneous biopsy (NCT04403607). Small resistance arteries were isolated and examined using wire myography, histopathology, immunohistochemistry, and spatial transcriptomics. Endothelium-independent (sodium nitroprusside) and -dependent (acetylcholine) vasorelaxation and vasoconstriction to the thromboxane A2 receptor agonist, U46619, and endothelin-1 (ET-1) in the presence or absence of a RhoA/Rho-kinase inhibitor (fasudil), were investigated. Thirty-seven patients, including 27 (mean age 57 years, 48% women, 41% cardiovascular disease) 3 months post-COVID-19 and 10 controls (mean age 57 years, 20% women, 30% cardiovascular disease), were included. Compared with control responses, U46619-induced constriction was increased (P = 0.002) and endothelium-independent vasorelaxation was reduced in arteries from COVID-19 patients (P < 0.001). This difference was abolished by fasudil. Histopathology revealed greater collagen abundance in COVID-19 arteries {Masson's trichrome (MT) 69.7% [95% confidence interval (CI): 67.8–71.7]; picrosirius red 68.6% [95% CI: 64.4–72.8]} vs. controls [MT 64.9% (95% CI: 59.4–70.3) (P = 0.028); picrosirius red 60.1% (95% CI: 55.4–64.8), (P = 0.029)]. Greater phosphorylated myosin light chain antibody-positive staining in vascular smooth muscle cells was observed in COVID-19 arteries (40.1%; 95% CI: 30.9–49.3) vs. controls (10.0%; 95% CI: 4.4–15.6) (P < 0.001). In proof-of-concept studies, gene pathways associated with extracellular matrix alteration, proteoglycan synthesis, and viral mRNA replication appeared to be upregulated. Conclusion Patients with post-COVID-19 conditions have enhanced vascular fibrosis and myosin light change phosphorylation. Rho-kinase activation represents a novel therapeutic target for clinical trials.

[1]  Christopher J. L. Murray,et al.  Estimated Global Proportions of Individuals With Persistent Fatigue, Cognitive, and Respiratory Symptom Clusters Following Symptomatic COVID-19 in 2020 and 2021. , 2022, JAMA.

[2]  P. Macfarlane,et al.  A multisystem, cardio-renal investigation of post-COVID-19 illness , 2022, Nature medicine.

[3]  Ying Sun,et al.  The SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells: Implications for COVID-19 vascular research , 2022, Stem Cell Reports.

[4]  A. Gasbarrini,et al.  Impaired Endothelial Function in Convalescent Phase of COVID-19: A 3 Month Follow Up Observational Prospective Study , 2022, Journal of clinical medicine.

[5]  C. Tsioufis,et al.  Endothelial dysfunction in acute and long standing COVID−19: A prospective cohort study , 2022, Vascular Pharmacology.

[6]  M. Radisic,et al.  Cardiovascular signatures of COVID-19 predict mortality and identify barrier stabilizing therapies , 2022, eBioMedicine.

[7]  L. Poon,et al.  A human pluripotent stem cell-based model of SARS-CoV-2 infection reveals an ACE2-independent inflammatory activation of vascular endothelial cells through TLR4 , 2022, Stem Cell Reports.

[8]  M. Aepfelbacher,et al.  Multi-organ assessment in mainly non-hospitalized individuals after SARS-CoV-2 infection: The Hamburg City Health Study COVID programme , 2022, European heart journal.

[9]  M. Lederman,et al.  SARS-CoV-2 Spike Protein Destabilizes Microvascular Homeostasis , 2021, Microbiology spectrum.

[10]  Y. Nishijima,et al.  Prolonged endothelial-dysfunction in human arterioles following infection with SARS-CoV-2 , 2021, Cardiovascular research.

[11]  H. Jha,et al.  Repurposing of gastric cancer drugs against COVID-19 , 2021, Computers in Biology and Medicine.

[12]  T. Renné,et al.  Persistent endotheliopathy in the pathogenesis of long COVID syndrome , 2021, Journal of Thrombosis and Haemostasis.

[13]  E. Poveda,et al.  Long COVID in hospitalized and non-hospitalized patients in a large cohort in Northwest Spain, a prospective cohort study , 2021, Scientific Reports.

[14]  J. Chase,et al.  Post-intensive care syndrome after a critical COVID-19: cohort study from a Belgian follow-up clinic , 2021, Annals of Intensive Care.

[15]  Robert A. Campbell,et al.  COVID-19 generates hyaluronan fragments that directly induce endothelial barrier dysfunction , 2021, JCI insight.

[16]  P. Fadel,et al.  Blunted peripheral but not cerebral vasodilator function in young otherwise healthy adults with persistent symptoms following COVID-19 , 2021, American journal of physiology. Heart and circulatory physiology.

[17]  C. Dieterich,et al.  Increased susceptibility of human endothelial cells to infections by SARS-CoV-2 variants , 2021, Basic Research in Cardiology.

[18]  H. Sethi,et al.  Ripasudil Endgame: Role of Rho-Kinase Inhibitor as a Last-Ditch-Stand Towards Maximally Tolerated Medical Therapy to a Patient of Advanced Glaucoma , 2021, Clinical ophthalmology.

[19]  M. Maniscalco,et al.  Clinical Assessment of Endothelial Function in Convalescent COVID-19 Patients Undergoing Multidisciplinary Pulmonary Rehabilitation , 2021, Biomedicines.

[20]  Kevin N. Heath,et al.  Risk of clinical sequelae after the acute phase of SARS-CoV-2 infection: retrospective cohort study , 2021, BMJ.

[21]  W. Kuebler,et al.  In vitro screening identifies TRPV4 as target for endothelial barrier stabilization in COVID‐19 , 2021, The FASEB Journal.

[22]  A. Malhotra,et al.  SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2 , 2021, Circulation research.

[23]  J. Haas,et al.  Lack of Evidence of Angiotensin-Converting Enzyme 2 Expression and Replicative Infection by SARS-CoV-2 in Human Endothelial Cells , 2021, Circulation.

[24]  P. Libby,et al.  COVID-19 is, in the end, an endothelial disease , 2020, European heart journal.

[25]  P. Macfarlane,et al.  The Chief Scientist Office Cardiovascular and Pulmonary Imaging in SARS Coronavirus disease-19 (CISCO-19) study , 2020, Cardiovascular research.

[26]  L. Calò,et al.  Letter: ACE2, Rho kinase inhibition and the potential role of vitamin D against COVID‐19 , 2020, Alimentary pharmacology & therapeutics.

[27]  P. Davis,et al.  Rho kinase inhibitors for SARS-CoV-2 induced acute respiratory distress syndrome: Support from Bartter’s and Gitelman’s syndrome patients , 2020, Pharmacological Research.

[28]  G. Karimi,et al.  Plausibility of therapeutic effects of Rho kinase inhibitors against Severe Acute Respiratory Syndrome Coronavirus 2 (COVID-19) , 2020, Pharmacological Research.

[29]  G. Herrler,et al.  SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor , 2020, Cell.

[30]  R. Touyz,et al.  Crosstalk Between Vascular Redox and Calcium Signaling in Hypertension Involves TRPM2 (Transient Receptor Potential Melastatin 2) Cation Channel. , 2019, Hypertension.

[31]  P. Karki,et al.  Rho and reactive oxygen species at crossroads of endothelial permeability and inflammation. , 2019, Antioxidants & redox signaling.

[32]  C. Berry,et al.  Systemic microvascular dysfunction in microvascular and vasospastic angina , 2018, European heart journal.

[33]  R. Touyz,et al.  Vascular smooth muscle contraction in hypertension , 2018, Cardiovascular research.

[34]  S. Boland,et al.  Rho kinase inhibitors: a patent review (2014 – 2016) , 2017, Expert opinion on therapeutic patents.

[35]  C. Solomon,et al.  The endothelial glycocalyx and its disruption, protection and regeneration: a narrative review , 2016, Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine.

[36]  H. Shimokawa,et al.  RhoA/Rho-Kinase in the Cardiovascular System. , 2016, Circulation research.

[37]  W. Gasper,et al.  Ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery in clinical research. , 2014, Journal of visualized experiments : JoVE.

[38]  P. Lograsso,et al.  Rho kinase inhibitors: a patent review (2012 – 2013) , 2014, Expert opinion on therapeutic patents.

[39]  Yongbo Zhang,et al.  Rho kinase: A new target for treatment of cerebral ischemia/reperfusion injury , 2013, Neural regeneration research.

[40]  B. M. Saraiva-Romanholo,et al.  Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. , 2012, American journal of physiology. Lung cellular and molecular physiology.

[41]  A. D'Angelo,et al.  ACE2 and angiotensin 1-7 are increased in a human model of cardiovascular hyporeactivity: pathophysiological implications. , 2010, Journal of nephrology.

[42]  R. Shenkar,et al.  Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity , 2010, The Journal of experimental medicine.

[43]  V. Videm,et al.  Soluble ICAM‐1 and VCAM‐1 as Markers of Endothelial Activation , 2008, Scandinavian journal of immunology.

[44]  F Verrecchia,et al.  [Cellular and molecular mechanisms of fibrosis]. , 2006, Annales de pathologie.

[45]  Julie H. Campbell,et al.  Rho and vascular disease. , 2005, Atherosclerosis.

[46]  A. Takeshita,et al.  Inflammatory stimuli upregulate Rho-kinase in human coronary vascular smooth muscle cells. , 2004, Journal of molecular and cellular cardiology.

[47]  G. Navis,et al.  Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis , 2004, The Journal of pathology.

[48]  S. Bolz,et al.  Nitric Oxide‐Induced Decrease in Calcium Sensitivity of Resistance Arteries Is Attributable to Activation of the Myosin Light Chain Phosphatase and Antagonized by the RhoA/Rho Kinase Pathway , 2003, Circulation.

[49]  K. Kaibuchi,et al.  Involvement of Rho-kinase-mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm. , 2000, Circulation research.

[50]  A. Bloom.  The biosynthesis of factor VIII. , 1979, Clinics in haematology.