sj-pdf-1-ajs-10.1177_03635465231195850 – Supplemental material for Therapeutic Myogenesis Induced by Ultrasound Exposure in a Volumetric Skeletal Muscle Loss Injury Model

Supplemental material, sj-pdf-1-ajs-10.1177_03635465231195850 for Therapeutic Myogenesis Induced by Ultrasound Exposure in a Volumetric Skeletal Muscle Loss Injury Model by Farina Mohamad Yusoff, Ayumu Nakashima, Masato Kajikawa, Shinji Kishimoto, Tatsuya Maruhashi and Yukihito Higashi in The American Journal of Sports Medicine</p

Clinical characteristics of subjects.

The effects of Covid-19 vaccines on vascular function are still controversial. We evaluated the effects of BNT162b2 vaccine (BioNTech and Pfizer) on endothelial function assessed by flow-mediated vasodilation (FMD) and vascular smooth muscle function assessed by nitroglycerine-induced vasodilation (NID). This study was a prospective observational study. A total of 23 medical staff at Hiroshima University Hospital were enrolled in this study. FMD and NID were measured before vaccination and two weeks and six months after the 2nd dose of vaccination. FMD was significantly smaller two weeks after the 2nd dose of vaccination than before vaccination (6.5±2.4% and 8.2±2.6%, p = 0.03). FMD was significantly larger at six months than at two weeks after the 2nd dose of vaccination (8.2±3.0% and 6.5±2.4%, p = 0.03). There was no significant difference between FMD before vaccination and that at six months after the 2nd dose of vaccination (8.2±2.6% to 8.2±3.0%, p = 0.96). NID values were similar before vaccination and at two weeks, and six months after vaccination (p = 0.89). The BNT162b2 Covid-19 vaccine temporally impaired endothelial function but not vascular smooth muscle function, and the impaired endothelial function returned to the baseline level within six months after vaccination.</div

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The effects of Covid-19 vaccines on vascular function are still controversial. We evaluated the effects of BNT162b2 vaccine (BioNTech and Pfizer) on endothelial function assessed by flow-mediated vasodilation (FMD) and vascular smooth muscle function assessed by nitroglycerine-induced vasodilation (NID). This study was a prospective observational study. A total of 23 medical staff at Hiroshima University Hospital were enrolled in this study. FMD and NID were measured before vaccination and two weeks and six months after the 2nd dose of vaccination. FMD was significantly smaller two weeks after the 2nd dose of vaccination than before vaccination (6.5±2.4% and 8.2±2.6%, p = 0.03). FMD was significantly larger at six months than at two weeks after the 2nd dose of vaccination (8.2±3.0% and 6.5±2.4%, p = 0.03). There was no significant difference between FMD before vaccination and that at six months after the 2nd dose of vaccination (8.2±2.6% to 8.2±3.0%, p = 0.96). NID values were similar before vaccination and at two weeks, and six months after vaccination (p = 0.89). The BNT162b2 Covid-19 vaccine temporally impaired endothelial function but not vascular smooth muscle function, and the impaired endothelial function returned to the baseline level within six months after vaccination.</div

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Dot graphs show effects of the BNT162b2 mRNA Covid-19 vaccine on flow-mediated vasodilation (A) and nitroglycerine-induced vasodilation (B) before vaccination and at two weeks and six months after the 2nd dose of vaccination.</p

Body Weight, Systemic Hemodynamics and Heart Weight/Tibial Length in Control, Ang II-infused, SNP-infused, and A combined Ang II and SNP-infused Mice.

<p>Ang II indicates angiotensin II; SNP, sodium nitroprusside.</p><p>*P<0.05 vs. before,</p>†<p>P<0.05 vs. control.</p><p>All results are presented as mean±SD.</p><p>Body Weight, Systemic Hemodynamics and Heart Weight/Tibial Length in Control, Ang II-infused, SNP-infused, and A combined Ang II and SNP-infused Mice.</p

Effect of SNP on Ang II-induced ROCK activation in mouse aortas.

<p>Mice were treated with saline or Ang II via implanted micro-osmotic pump infusion (2 mg/kg/day) and treated with saline or SNP via subcutaneous injection (5 mg/kg once a day) for 14 days. Increased blood pressures in Ang II-treated mice were normalized to levels similar to those in mice without Ang II treatment by hydralazine included in drinking water (20 mg/kg/day). <b>(A)</b> Representative results of Western blot analysis for p-MBS, t-MBS, ROCK1, ROCK2, and actin. <b>(B)</b> Quantitative analysis of relative ROCK activity (p-MBS/t-MBS). <b>(C)</b> Quantitative analysis of relative ROCK1 expression. <b>(D)</b> Quantitative analysis of relative ROCK2 expression. n = 5–11 in each group; **P<0.01 compared to control; ^††P<0.01 compared to Ang II.</p

Effect of SNP on Ang II-induced ROCK activation in human aortic VSMCs.

<p>Human aortic VSMCs were treated with a vehicle or SNP (40 µmol/L) for 3 hours and stimulated with saline or Ang II (40 µmol/L) for the last 1 hour. <b>(A)</b> Representative results of Western blot analysis for p-MBS, t-MBS, ROCK1, ROCK2, and actin. <b>(B)</b> Quantitative analysis of relative ROCK activity (p-MBS/t-MBS). <b>(C)</b> Quantitative analysis of relative ROCK1 expression. <b>(D)</b> Quantitative analysis of relative ROCK2 expression. n = 4 in each group. *P<0.05 compared to control; ^††P<0.01 compared to Ang II. ^†P<0.05 compared to Ang II.</p

Effect of SNP on cGMP accumulation and cell migration in murine and human aortic VSMCs.

<p><b>(A)</b> cGMP accumulation was evaluated after treatment with a vehicle or SNP (40 µmol/L) for 5 minutes in murine VSMCs. n = 6 to 8 in each group. <b>(B)</b> Murine VSMCs migration was evaluated using a modified Boyden chamber assay after 24 hours of incubation with a vehicle or SNP (40 µmol/L) and/or Ang II (40 µmol/L). n = 12 to 13 in each group. <b>(C)</b> cGMP accumulation was evaluated after treatment with a vehicle or SNP (40 µmol/L) for 5 minutes in human aortic VSMCs. n = 5 to 7 in each group. <b>(D)</b> Human aortic VSMCs migration was evaluated using a modified Boyden chamber assay after 24 hours of incubation with a vehicle or SNP (40 µmol/L) and/or Ang II (40 µmol/L). n = 12 in each group. *P<0.05 compared to control; ^§P<0.0001 compared to control; ^§§P<0.0001 compared to Ang II. **P<0.01 compared to control.</p

Angiographic Score after Implantation of MSCs.

<p>(A and B) Representative angiography at 28 days after MSC implantation or saline injection (control) in rabbits with limb ischemia, (C) Angiographic score at 28 days after MSC implantation or saline injection (control) in rabbits with limb ischemia. *P<0.05 vs. the control group.</p

LDPI after Implantation of MSCs.

<p>(A and B) Representative laser LDPIs before and at 1, 7 and 28 days after MSC implantation or saline injection (control) in rabbits with limb ischemia, (C) High perfusion is indicated by red, and low perfusion is indicated by blue. Time-dependent change in LDPI index at 1, 7 and 28 days after MSC implantation or saline injection (control).</p