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

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.

Osteoprotegerin regulates vascular function through syndecan-1 and NADPH oxidase-derived reactive oxygen species.

Osteogenic factors, such as osteoprotegerin (OPG), are protective against vascular calcification. However, OPG is also positively associated with cardiovascular damage, particularly in pulmonary hypertension, possibly through processes beyond effects on calcification. In the present study, we focused on calcification-independent vascular effects of OPG through activation of syndecan-1 and NADPH oxidases (Noxs) 1 and 4. Isolated resistance arteries from Wistar-Kyoto (WKY) rats, exposed to exogenous OPG, studied by myography exhibited endothelial and smooth muscle dysfunction. OPG decreased nitric oxide (NO) production, eNOS activation and increased reactive oxygen species (ROS) production in endothelial cells. In VSMCs, OPG increased ROS production, H2O2/peroxynitrite levels and activation of Rho kinase and myosin light chain. OPG vascular and redox effects were also inhibited by the syndecan-1 inhibitor synstatin (SSNT). Additionally, heparinase and chondroitinase abolished OPG effects on VSMCs-ROS production, confirming syndecan-1 as OPG molecular partner and suggesting that OPG binds to heparan/chondroitin sulphate chains of syndecan-1. OPG-induced ROS production was abrogated by NoxA1ds (Nox1 inhibitor) and GKT137831 (dual Nox1/Nox4 inhibitor). Tempol (SOD mimetic) inhibited vascular dysfunction induced by OPG. In addition, we studied arteries from Nox1 and Nox4 knockout (KO) mice. Nox1 and Nox4 KO abrogated OPG-induced vascular dysfunction. Vascular dysfunction elicited by OPG is mediated by a complex signalling cascade involving syndecan-1, Nox1 and Nox4. Our data identify novel molecular mechanisms beyond calcification for OPG, which may underlie vascular injurious effects of osteogenic factors in conditions such as hypertension and/or diabetes.

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

Identification and characterization of small-molecule ligands that maintain pluripotency of human embryonic stem cells.

hESCs (human embryonic stem cells) offer great potential for pharmaceutical research and development and, potentially, for therapeutic use. However, improvements in cell culture are urgently required to allow the scalable production of large numbers of cells that maintain pluripotency. Supplementing feeder-free conditions with either EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine] or readily synthesized analogues of this compound maintains hESC pluripotency in the absence of exogenous cytokines. When the hESC lines SA121 or SA461 were maintained in feeder-free conditions with EHNA they displayed no reduction in stem-cell-associated markers such as Nanog, Oct4 (octamer-binding protein 4) and SSEA4 (stage-specific embryonic antigen 4) when compared with cells maintained in full feeder-free conditions that included exogenously added bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but EHNA did not limit efficient spontaneous or directed differentiation following its removal. We conclude that EHNA or related compounds offers a viable alternative to exogenous cytokine addition in maintaining hESC cultures in a pluripotent state and might be a particularly useful replacement for bFGF for large-scale or GMP (good manufacturing practice)-compliant processes.

Inherited CAG.CTG allele length is a major modifier of somatic mutation length variability in Huntington disease.

Huntington disease (HD) is associated with an unstable trinucleotide CAG.CTG repeat expansion. Although the repeat length is inversely correlated with the age-at-onset of symptoms, variability between patients who have inherited the same HD repeat length clearly suggests that other factors influence this aspect of the disease. As repeat length profiles in somatic tissues suggest that repeat length gains may contribute to both the tissue-specificity and progressive nature of HD pathogenesis, genetic modifiers of mutation length variability may therefore influence the age-at-onset of the disease. Using a sensitive single molecule-PCR assay we show that HD mutation length profiles in buccal cell DNA vary from individual to individual. The resulting data provide the first quantitative evidence that inherited CAG.CTG repeat length has a major influence on somatic CAG.CTG repeat length variation. In addition, we confirm that further environmental and/or genetic modifiers of repeat length variation exist and discuss the implications that our results may have on understanding the factors that influence severity and age-at-onset of Huntington disease symptoms.

Increased SK3 expression in DM1 lens cells leads to impaired growth through a greater calcium-induced fragility.

Although cataract is a characteristic feature of myotonic dystrophy type 1 (DM1), little is known of the underlying mechanisms. We generated four lens epithelial cell lines derived from DM1 cataracts and two from age-matched, non-DM cataracts. Small-pool PCR revealed typical large triplet repeat expansions in the DM1 cells. Furthermore, real-time PCR analysis showed reduced SIX5 expression and increased expression of the Ca(2+)-activated K(+) channel SK3 in the DM1 cells. These cells also exhibited longer population doubling times which did not arise through reduced proliferation, but rather increased cell death as shown by increased release of lactate dehydrogenase (LDH). Using (86)Rb(+) as a tracer for K(+), we found no difference in the resting K(+) influx or efflux kinetics. In all cases, the ouabain sensitive component of the influx contributed approximately 50% of the total. However, stimulating internal Ca(2+) by exposure to ionomycin not only caused greater stimulation of K(+) ((86)Rb) efflux in the DM1 cells but also induced a higher rate of cell death (LDH assay). Since both the hyper-stimulation of K(+) efflux and cell death were reduced by the highly specific SK inhibitor apamin, we suggest that increased expression of SK3 has a critical role in the increased Ca(2+)-induced fragility in DM1 cells. The present data, therefore, both help explain the lower epithelial cell density previously observed in DM1 cataracts and provide general insights into mechanisms underlying the fragility of other DM1-affected tissues.

Pms2 is a genetic enhancer of trinucleotide CAG.CTG repeat somatic mosaicism: implications for the mechanism of triplet repeat expansion.

The expansion of CAG.CTG repeat sequences is the cause of several inherited human disorders. Longer alleles are associated with an earlier age of onset and more severe symptoms, and are highly unstable in the germline and soma with a marked tendency towards repeat length gains. Germinal expansions underlie anticipation; whereas age-dependent, tissue-specific, expansion-biased somatic instability probably contributes toward the progressive nature and tissue-specificity of the symptoms. The mechanism(s) of repeat instability is not known, but recent data have implicated mismatch-repair (MMR) gene mutS homologues in driving expansion. To gain further insight into the expansion mechanism, we have determined the levels of somatic mosaicism of a transgenic expanded CAG.CTG repeat in mice deficient for the Pms2 MMR gene. Pms2 is a MutL homologue that plays a critical role in the downstream processing of DNA mismatches. The rate of somatic expansion was reduced by approximately 50% in Pms2-null mice. A higher frequency of rare, but very large, deletions was also detected in these animals. No significant differences were observed between Pms2(+/+) and Pms2(+/-) mice, indicating that a single functional Pms2 allele is sufficient to generate normal levels of somatic mosaicism. These findings reveal that as well as MMR enzymes that directly bind mismatched DNA, proteins that are subsequently recruited to the complex also play a central role in the accumulation of repeat length changes. These data suggest that somatic expansion results not by replication slippage, single stranded annealing or simple MutS-mediated stabilization of secondary structures, but by inappropriate DNA MMR.

Genomic context drives SCA7 CAG repeat instability, while expressed SCA7 cDNAs are intergenerationally and somatically stable in transgenic mice.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant cerebellar ataxia caused by a CAG repeat expansion in the ataxin-7 gene. In humans, SCA7 is characterized by marked anticipation due to intergenerational repeat instability with a bias toward expansion, and is thus regarded as the most unstable of the polyglutamine diseases. To study the molecular basis of CAG/CTG repeat instability and its pathological significance, we generated lines of transgenic mice carrying either a SCA7 cDNA construct or a 13.5 kb SCA7 genomic fragment with 92 CAG repeats. While the cDNA transgenic mice showed little intergenerational repeat instability, the genomic fragment transgenic mice displayed marked intergenerational instability with an obvious expansion bias. We then went on to generate additional lines of genomic fragment transgenic mice, and observed that deletion of the 3' genomic region significantly stabilized intergenerational transmission of the SCA7 CAG92 repeat. These results suggest that cis-information present on the genomic fragment is driving the instability process. As the SCA7 genomic fragment contains a large number of replication-associated motifs, the presence of such sequence elements may make the SCA7 CAG repeat region more susceptible to instability. Small-pool and standard PCR analysis of tissues from genomic fragment mice revealed large repeat expansions in their brains and livers, but no such changes were found in any tissues from cDNA transgenic mice that have been shown to undergo neurodegeneration. As large somatic repeat expansions are absent from the brains of SCA7 cDNA mice, our results indicate that neurodegeneration can occur without marked somatic mosaicism, at least in these mice.

Instability of a premutation allele in homozygous patients with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is caused by the expansion of an unstable CTG repeat in the DMPK gene on chromosome 19q13.3. We present two siblings with DM1 who each inherited a premutation allele, (CTG)43, stably transmitted from the mother and a full-mutation allele, either (CTG)500 or (CTG)180, derived from a paternal protomutation allele, (CTG)52. Small-pool polymerase chain reaction analysis showed that the (CTG)52 repeat allele was relatively stable in somatic tissues but was highly unstable in the male germline and extremely biased toward further expansion, consistent with the high levels of anticipation observed in DM1 families. The (CTG)43 allele showed subtle somatic instability in the mother, with maximum additions of two repeats and deletions of one repeat. Conversely, in the younger affected siblings the (CTG)43 allele showed a high degree of somatic instability (approximately 70% mutation load), resulting in deletions reverting to the high end of the normal range (down to [CTG]33) and additions up to the proto-mutation range (up to [CTG]64). The difference in the somatic stability of the (CTG)43 allele between the mother and her offspring suggests that interallelic interactions or other mechanisms in trans regulate the stability of the (CTG)43 premutation allele.