Vascular mechanisms of post-COVID-19 conditions: rho-kinase is a novel target for therapy 2023 Sykes et al

[Andy]

Background
In post-COVID-19 conditions (Long COVID), systemic vascular dysfunction is implicated but the mechanisms are uncertain, and treatment is imprecise.

Methods
Patients convalescing after hospitalisation 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.

Results
Thirty-seven patients, including 27 (mean age 57 years, 48% women, 41% cardiovascular disease) three 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%CI: 67.8, 71.7]; picrosirius red 68.6% [95% CI: 64.4, 72.8]) versus 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.

Open access, https://academic.oup.com/ehjcvp/advance-article/doi/10.1093/ehjcvp/pvad025/7109259

 

[SNT Gatchaman]

These sound like patients with significant acute disease and prior co-morbidities, so uncertain how well this relates to the larger numbers of LC patients with mild or asymptomatic acute infection, particularly those who were previously healthy.

We undertook a prospective, observational, multicentre, secondary care cohort study assessing the prevalence and clinical significance of multi-organ injury in survivors of COVID-19 during convalescence.

Thirty-seven patients, including twenty-seven (mean age 57 years, 48% women, 41% cardiovascular disease) with persisting cardiovascular symptoms three months after hospitalisation for COVID-19 and ten controls (mean age 57 years, 20% women, 30% cardiovascular disease), were prospectively included. The control patients had received hospital-based care either as inpatients or outpatients.

Of cases: 93% hospitalised; median admission 10 days; 33% in ICU, 19% with invasive ventilation, 11% on inotropes; 48% former or current smokers; 7% previous MI; 7% previous stroke; 96% angina.

 

[SNT Gatchaman]

Also peak ferritin highlighted in Table 1, with median/IQR —

Controls: 152 (97-188) µg/L
Cases: 327 (200-1505) µg/L
p: 0.018

 

[Hutan]

Yes, hospitalised post-Covid-19 samples, poorly matched controls with respect to sex and exposure to Covid-19 and only 10 controls.

On ET-1

Biomarkers of vascular inflammation including ET-1, ICAM-1, and VCAM-1 with factor VIII, protein C, and vWF activity were increased at enrolment, although returned to comparable levels with controls within 60 days of discharge from hospital, suggesting that persistent acute inflammation is not responsible for post-COVID-19

 

[Hutan]

We hypothesized that SARS-CoV-2 infection impairs non-endothelium-dependent vasorelaxation pathways through VSMC and RhoA/Rho-kinase activation, which, together with altered calcium ion (Ca2+) handling in these cells, impairs vascular function in patients with post-COVID-19 conditions, compared with matched controls.

They are suggesting that issues with vascular function, specifically constriction or relaxation, might be a reason for post-Covid symptoms. The questions are interesting, and there might be some results of interest to us here. It's just that there is so many confounding factors and the sample sizes are small.

 

[Hutan]

Compared to contractile responses to U46619, the thromboxane A2 agonist, in small arteries isolated from control patients, the contractile responses in small arteries isolated from post-COVID-19 patients were increased (P = 0.039) (Figure 1A). SNP-induced vasorelaxation (endothelium-independent) was significantly reduced relative to controls (P = 0.012) (Figure 1B). There were no between-group differences in acetylcholine (ACh)-induced vasorelaxation (endothelium-dependent) (P = 0.880) (Figure 1C) or contractile responses to ET-1 (P = 0.631) (Figure 1D).

In subsequent studies, selective pre-treatment of the small arteries with fasudil, a potent inhibitor of the Rho-kinases ROCK1 and ROCK2, restored SNP-mediated vasodilatation and U46619 hypercontractile response (Figure 1A, B). Fasudil did not change SNP and U46619 responses in small arteries from control patients

Immunohistochemical analysis (Figure 2) was undertaken to investigate the downstream effects of Rho-kinase in this cohort, with staining for phosphorylated myosin light chain (pMLC) antibodies (Abcam ab2480). pMLC phosphorylation reflects enhanced sensitivity towards Ca2+ linking actin–myosin filaments for VSMC contraction. Overall, much greater pMLC antibody positive tissue was observed in the COVID-19 vessels at ×40.0 magnification, using a total slide tissue proportion threshold analysis technique, COVID-19 (40.1%; 95% CI: 30.9–49.3; n = 21) vs. controls (10.0%; 95% CI: 4.4–15.6; n = 9) P < 0.001. This mechanism for Rho-kinase inhibition, through a reduction in MLC activity, corroborates the mediation of hypercontractility and impaired vasodilation observed during wire myography experiments.

Figure 2
Box and whisker plot of COVID-19 (red) and age, sex, and cardiovascular risk factor matched controls (blue) immunohistochemical staining for phosphorylated myosin light chain antibody uptake. Increased mean proportion of staining for phosphorylated myosin light chain antibody uptake is observed in patients following COVID-19 (n = 21) (40.1%; 95% CI: 30.9–49.3) compared with age, sex, and cardiovascular risk factor matched controls (n = 9) (10.0%; 95% CI: 4.4–15.6) (P < 0.001). Illustrative example images are provided demonstrating the ×40.0 magnification with 60 uM scale bar.