Reconstituted Extracellular Vesicles from Human Platelets Decrease Viral Myocarditis in Mice.

Patients with viral myocarditis are at risk of sudden death and may progress to dilated cardiomyopathy (DCM). Currently, no disease-specific therapies exist to treat viral myocarditis. Here it is examined whether reconstituted, lyophilized extracellular vesicles (EVs) from platelets from healthy men and women reduce acute or chronic myocarditis in male mice. Human-platelet-derived EVs (PEV) do not cause toxicity, damage, or inflammation in naïve mice. PEV administered during the innate immune response significantly reduces myocarditis with fewer epidermal growth factor (EGF)-like module-containing mucin-like hormone receptor-like 1 (F4/80) macrophages, T cells (cluster of differentiation molecules 4 and 8, CD4 and CD8), and mast cells, and improved cardiac function. Innate immune mediators known to increase myocarditis are decreased by innate PEV treatment including Toll-like receptor (TLR)4 and complement. PEV also significantly reduces perivascular fibrosis and remodeling including interleukin 1 beta (IL-1ß), transforming growth factor-beta 1, matrix metalloproteinase, collagen genes, and mast cell degranulation. PEV given at days 7-9 after infection reduces myocarditis and improves cardiac function. MicroRNA (miR) sequencing reveals that PEV contains miRs that decrease viral replication, TLR4 signaling, and T-cell activation. These data show that EVs from the platelets of healthy individuals can significantly reduce myocarditis and improve cardiac function.

High overlap in patients diagnosed with hypermobile Ehlers-Danlos syndrome or hypermobile spectrum disorders with fibromyalgia and 40 self-reported symptoms and comorbidities.

Background: Joint pain is a common symptom in patients with hypermobile Ehlers-Danlos Syndrome (hEDS), hypermobility spectrum disorders (HSD) and fibromyalgia. The goal of this study was to determine whether symptoms and comorbidities overlap in patients diagnosed with hEDS/HSD and/or fibromyalgia. Methods: We retrospectively examined self-reported data from an EDS Clinic intake questionnaire in patients diagnosed with hEDS/HSD, fibromyalgia, or both vs. controls with an emphasis on joint issues. Results: From 733 patients seen at the EDS Clinic, 56.5% (n = 414) were diagnosed with hEDS/HSD and fibromyalgia (Fibro), 23.8% (n = 167) hEDS/HSD, 13.3% (n = 98) fibromyalgia, or 7.4% (n = 54) none of these diagnoses. More patients were diagnosed with HSD (76.6%) than hEDS (23.4%). Patients were primarily White (95%) and female (90%) with a median age in their 30s (controls 36.7 [18.0, 70.0], fibromyalgia 39.7 [18.0, 75.0], hEDS/HSD 35.0 [18.0, 71.0], hEDS/HSD&Fibro 31.0 [18.0, 63.0]). There was high overlap in all 40 symptoms/comorbidities that we examined in patients diagnosed with fibromyalgia only or hEDS/HSD&Fibro, regardless of whether they had hEDS or HSD. Patients that only had hEDS/HSD without fibromyalgia had far fewer symptoms/comorbidities than patients with hEDS/HSD&Fibro. The top self-reported issues in patients that only had fibromyalgia were joint pain, hand pain when writing or typing, brain fog, joint pain keeping from daily activities, allergy/atopy and headache. Five issues that significantly and uniquely characterized patients diagnosed with hEDS/HSD&Fibro were subluxations (dislocations in hEDS patients), joint issues like sprains, the need to stop sports due to injuries, poor wound healing, and migraine. Conclusion: The majority of patients seen at the EDS Clinic had a diagnosis of hEDS/HSD plus fibromyalgia that was associated with more severe disease. Our findings indicate that fibromyalgia should be routinely assessed in patients with hEDS/HSD and vis-a-versa to improve patient care.

Extracellular vesicles as personalized medicine.

Extracellular vesicles (EVs) are released from all cells in the body, forming an important intercellular communication network that contributes to health and disease. The contents of EVs are cell source-specific, inducing distinct signaling responses in recipient cells. The specificity of EVs and their accumulation in fluid spaces that are accessible for liquid biopsies make them highly attractive as potential biomarkers and therapies for disease. The duality of EVs as favorable (therapeutic) or unfavorable (pathological) messengers is context dependent and remains to be fully determined in homeostasis and various disease states. This review describes the use of EVs as biomarkers, drug delivery vehicles, and regenerative therapeutics, highlighting examples involving viral infections, cancer, and neurological diseases. There is growing interest to provide personalized therapy based on individual patient and disease characteristics. Increasing evidence suggests that EV biomarkers and therapeutic approaches are ideal for personalized medicine due to the diversity and multifunctionality of EVs.

Cardiovascular Factors Associated with COVID-19 from an International Registry of Primarily Japanese Patients.

Aims: We developed an international registry to examine cardiovascular complications of COVID-19. Methods: A REDCap form was created in March 2020 at Mayo Clinic in collaboration with the International Society of Cardiomyopathy, Myocarditis and Heart Failure (ISCMF) and data were entered from April 2020 through April 2021. Results: Of the 696 patients in the COVID-19 Registry, 411 (59.2%) were male and 283 (40.8%) were female, with a sex ratio of 1.5:1 male to female. In total, 95.5% of the patients were from Japan. The average age was 52 years with 31.5% being >65 years of age. COVID-19 patients with a history of cardiovascular disease (CVD) had more pre-existing conditions including type II diabetes (p < 0.0001), cancer (p = 0.0003), obesity (p = 0.001), and kidney disease (p = 0.001). They also had a greater mortality of 10.1% compared to 1.7% in those without a history of CVD (p < 0.0001). The most common cardiovascular conditions in patients with a history of CVD were hypertension (33.7%), stroke (5.7%) and arrhythmias (5.1%). We found that troponin T, troponin I, brain natriuretic peptide (BNP), N-terminal pro-BNP (NT-proBNP), C-reactive protein (CRP), IL-6 and lambda immunoglobulin free light chains (Ig FLC) were elevated above reference levels in patients with COVID-19. Myocarditis is known to occur mainly in adults under the age of 50, and when we examined biomarkers in patients that were ≤50 years of age and had no history of CVD we found that a majority of patients had elevated levels of troponin T (71.4%), IL-6 (59.5%), creatine kinase/CK-MB (57.1%), D-dimer (57.8%), kappa Ig FLC (75.0%), and lambda Ig FLC (71.4%) suggesting myocardial injury and possible myocarditis. Conclusions: We report the first findings to our knowledge of cardiovascular complications from COVID-19 in the first year of the pandemic in a predominantly Japanese population. Mortality was increased by a history of CVD and pre-existing conditions including type II diabetes, cancer, obesity, and kidney disease. Our findings indicate that even in cases where no abnormalities are found in ECG or ultrasound cardiography that myocardial damage may occur, and cardiovascular and inflammatory biomarkers may be useful for the diagnosis.

Sex differences in comorbidities associated with Sjögren's disease.

Background: Little is known about the association of comorbidities with sex and age at diagnosis in Sjögren's disease. We tested the hypothesis that sex differences occur in comorbidities in patients with Sjögren's disease. Methods: Patients with Sjögren's disease were identified from 11/1974 to 7/2018 in the Mayo Clinic electronic medical record and assessed for 22 comorbidities according to sex and age at diagnosis. Results: Of the 13,849 patients identified with Sjögren's disease, 11,969 (86%) were women and 1,880 (14%) men, primarily white (88%) with a sex ratio of 6.4:1 women to men. The mean age at diagnosis was 57 years for women and 59.7 years for men, and 5.6% had a diagnosis of fibromyalgia at Sjögren's diagnosis. Men with Sjögren's disease were more likely than women to be a current or past smoker. The average time to diagnosis of comorbidities after diagnosis of Sjögren's disease was 2.6 years. The top comorbidities in patients with Sjögren's disease were fibromyalgia (25%), depression (21.2%) and pain (16.4%). Comorbidities that occurred more often in women were hypermobile syndromes (31:1), CREST (29:1), migraine (23:1), Ehlers-Danlos syndrome (EDS) (22:1), Raynaud's syndrome (15:1), SLE (13:1), systemic sclerosis (SSc) (13:1), and fibromyalgia (12:1). Women with Sjögren's disease were at increased risk of developing hypermobile syndromes (RR 7.27, CI 1.00-52.71, p = 0.05), EDS (RR 4.43, CI 1.08-18.14, p = 0.039), CREST (RR 4.24, CI 1.56-11.50, p = 0.005), migraine (RR 3.67, CI 2.39-5.62, p < 0.001), fibromyalgia (RR 2.26, CI 1.92-2.66, p < 0.001), Raynaud's syndrome (RR 2.29, CI 1.77-2.96, p < 0.001), SLE (RR 2.13, CI 1.64-2.76, p < 0.001), and SSc (RR 2.05 CI 1.44-2.92; p < 0.001). In contrast, men with Sjögren's were at increased risk for developing myocardial infarction (RR 0.44, CI 0.35-0.55, p < 0.001), atherosclerosis/CAD (RR 0.44, CI 0.39-0.49, p < 0.001), cardiomyopathy (RR 0.63, CI 0.46-0.86, p = 0.003), stroke (RR 0.66 CI 0.51-0.85, p = 0.001), and congestive heart failure (RR 0.70, CI 0.57-0.85, p < 0.001). Conclusions: The top comorbidities in Sjögren's disease were fibromyalgia, depression, and pain. Women with Sjögren's disease had a higher relative risk of developing fibromyalgia, depression, pain, migraine, hypermobile syndrome, EDS and other rheumatic autoimmune diseases. Men with Sjögren's disease had higher risk of developing cardiovascular diseases.

Upregulation of Endothelin-1 May Predict Chemotherapy-Induced Cardiotoxicity in Women with Breast Cancer.

As survival in breast cancer patients from newer therapies increases, concerns for chemotherapy-induced cardiotoxicity (CIC) have offset some of these benefits, manifesting as a decline in left ventricular ejection fraction (LVEF). Patients receiving anthracycline-based chemotherapy followed by trastuzumab are at risk for CIC. Previous research evaluating whether clinical biomarkers predict cardiotoxicity has been inconsistent. Recently, angiotensin II type 1 receptor (ATR1) and endothelin 1 (ET1) have been shown to play a role in breast tumor growth. We evaluated ATR1 and ET1 expression in breast cancer tissue and its association with CIC. A total of 33 paraffin-embedded breast tissue specimens from women with breast cancer treated with anthracycline-based chemotherapy and trastuzumab were analyzed by immunohistochemistry (IHC) and qRT-PCR. We found that ET1 expression was increased in patients with an LVEF ≤ 50% (p = 0.032) with a lower LVEF correlating with higher ET1 expression (r = 0.377, p = 0.031). In patients with a change in LVEF of greater than 10%, greater ET1 expression was noted compared to those without a change in LVEF (p = 0.017). Increased ET1 expression in breast tumor tissue is associated with reduced LVEF. Future studies need to examine whether ET1 may be a tissue biomarker that helps predict the risk of developing CIC in women with breast cancer.

A Novel Circulating MicroRNA for the Detection of Acute Myocarditis.

BACKGROUND: The diagnosis of acute myocarditis typically requires either endomyocardial biopsy (which is invasive) or cardiovascular magnetic resonance imaging (which is not universally available). Additional approaches to diagnosis are desirable. We sought to identify a novel microRNA for the diagnosis of acute myocarditis. METHODS: To identify a microRNA specific for myocarditis, we performed microRNA microarray analyses and quantitative polymerase-chain-reaction (qPCR) assays in sorted CD4+ T cells and type 17 helper T (Th17) cells after inducing experimental autoimmune myocarditis or myocardial infarction in mice. We also performed qPCR in samples from coxsackievirus-induced myocarditis in mice. We then identified the human homologue for this microRNA and compared its expression in plasma obtained from patients with acute myocarditis with the expression in various controls. RESULTS: We confirmed that Th17 cells, which are characterized by the production of interleukin-17, are a characteristic feature of myocardial injury in the acute phase of myocarditis. The microRNA mmu-miR-721 was synthesized by Th17 cells and was present in the plasma of mice with acute autoimmune or viral myocarditis but not in those with acute myocardial infarction. The human homologue, designated hsa-miR-Chr8:96, was identified in four independent cohorts of patients with myocarditis. The area under the receiver-operating-characteristic curve for this novel microRNA for distinguishing patients with acute myocarditis from those with myocardial infarction was 0.927 (95% confidence interval, 0.879 to 0.975). The microRNA retained its diagnostic value in models after adjustment for age, sex, ejection fraction, and serum troponin level. CONCLUSIONS: After identifying a novel microRNA in mice and humans with myocarditis, we found that the human homologue (hsa-miR-Chr8:96) could be used to distinguish patients with myocarditis from those with myocardial infarction. (Funded by the Spanish Ministry of Science and Innovation and others.).

Use of convalescent plasma in COVID-19 patients with immunosuppression.

In the absence of effective countermeasures, human convalescent plasma has been widely used to treat severe acute respiratory syndrome coronavirus 2, the causative agent of novel coronavirus disease 19 (COVID-19), including among patients with innate or acquired immunosuppression. However, the association between COVID-19-associated mortality in patients with immunosuppression and therapeutic use of convalescent plasma is unknown. We review 75 reports, including one large matched-control registry study of 143 COVID-19 patients with hematological malignancies, and 51 case reports and 23 case series representing 238 COVID-19 patients with immunosuppression. We review clinical features and treatment protocols of COVID-19 patients with immunosuppression after treatment with human convalescent plasma. We also discuss the time course and clinical features of recovery. The available data from case reports and case series provide evidence suggesting a mortality benefit and rapid clinical improvement in patients with several forms of immunosuppression following COVID-19 convalescent plasma transfusion. The utility of convalescent plasma or other forms of antibody therapy in immune-deficient and immune-suppressed patients with COVID-19 warrants further investigation.

Trpc6 Promotes Doxorubicin-Induced Cardiomyopathy in Male Mice With Pleiotropic Differences Between Males and Females.

Background: Doxorubicin is a widely used and effective chemotherapy, but the major limiting side effect is cardiomyopathy which in some patients leads to congestive heart failure. Genetic variants in TRPC6 have been associated with the development of doxorubicin-induced cardiotoxicity, suggesting that TRPC6 may be a therapeutic target for cardioprotection in cancer patients. Methods: Assessment of Trpc6 deficiency to prevent doxorubicin-induced cardiac damage and function was conducted in male and female B6.129 and Trpc6 knock-out mice. Mice were treated with doxorubicin intraperitoneally every other day for a total of 6 injections (4 mg/kg/dose, cumulative dose 24 mg/kg). Cardiac damage was measured in heart sections by quantification of vacuolation and fibrosis, and in heart tissue by gene expression of Tnni3 and Myh7. Cardiac function was determined by echocardiography. Results: When treated with doxorubicin, male Trpc6-deficient mice showed improvement in markers of cardiac damage with significantly reduced vacuolation, fibrosis and Myh7 expression and increased Tnni3 expression in the heart compared to wild-type controls. Similarly, male Trpc6-deficient mice treated with doxorubicin had improved LVEF, fractional shortening, cardiac output and stroke volume. Female mice were less susceptible to doxorubicin-induced cardiac damage and functional changes than males, but Trpc6-deficient females had improved vacuolation with doxorubicin treatment. Sex differences were observed in wild-type and Trpc6-deficient mice in body-weight and expression of Trpc1, Trpc3 and Rcan1 in response to doxorubicin. Conclusions: Trpc6 promotes cardiac damage following treatment with doxorubicin resulting in cardiomyopathy in male mice. Female mice are less susceptible to cardiotoxicity with more robust ability to modulate other Trpc channels and Rcan1 expression.

Association of Genetic Variants at TRPC6 With Chemotherapy-Related Heart Failure.

Background: Our previous GWAS identified genetic variants at six novel loci that were associated with a decline in left ventricular ejection fraction (LVEF), p < 1 × 10-5 in 1,191 early breast cancer patients from the N9831 clinical trial of chemotherapy plus trastuzumab. In this study we sought replication of these loci. Methods: We tested the top loci from the GWAS for association with chemotherapy-related heart failure (CRHF) using 26 CRHF cases from N9831 and 984 patients from the Mayo Clinic Biobank which included CRHF cases (N = 12) and control groups of patients treated with anthracycline +/- trastuzumab without HF (N = 282) and patients with HF that were never treated with anthracycline or trastuzumab (N = 690). We further examined associated loci in the context of gene expression and rare coding variants using a TWAS approach in heart left ventricle and Sanger sequencing, respectively. Doxorubicin-induced apoptosis and cardiomyopathy was modeled in human iPSC-derived cardiomyocytes and endothelial cells and a mouse model, respectively, that were pre-treated with GsMTx-4, an inhibitor of TRPC6. Results: TRPC6 5' flanking variant rs57242572-T was significantly more frequent in cases compared to controls, p = 0.031, and rs61918162-T showed a trend for association, p = 0.065. The rs61918162 T-allele was associated with higher TRPC6 expression in the heart left ventricle. We identified a single TRPC6 rare missense variant (rs767086724, N338S, prevalence 0.0025% in GnomAD) in one of 38 patients (2.6%) with CRHF. Pre-treatment of cardiomyocytes and endothelial cells with GsMTx4 significantly reduced doxorubicin-induced apoptosis. Similarly, mice treated with GsMTx4 had significantly improved doxorubicin-induced cardiac dysfunction. Conclusions: Genetic variants that are associated with increased TRPC6 expression in the heart and rare TRPC6 missense variants may be clinically useful as risk factors for CRHF. GsMTx-4 may be a cardioprotective agent in patients with TRPC6 risk variants. Replication of the genetic associations in larger well-characterized samples and functional studies are required.

Therapeutic modulation of phagocytosis in glioblastoma can activate both innate and adaptive antitumour immunity.

Tumour cell phagocytosis by antigen presenting cells (APCs) is critical to the generation of antitumour immunity. However, cancer cells can evade phagocytosis by upregulating anti-phagocytosis molecule CD47. Here, we show that CD47 blockade alone is inefficient in stimulating glioma cell phagocytosis. However, combining CD47 blockade with temozolomide results in a significant pro-phagocytosis effect due to the latter's ability to induce endoplasmic reticulum stress response. Increased tumour cell phagocytosis subsequently enhances antigen cross-presentation and activation of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) in APCs, resulting in more efficient T cell priming. This bridging of innate and adaptive responses inhibits glioma growth, but also activates immune checkpoint. Sequential administration of an anti-PD1 antibody overcomes this potential adaptive resistance. Together, these findings reveal a dynamic relationship between innate and adaptive immune regulation in tumours and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

Elevated Sera sST2 Is Associated With Heart Failure in Men ≤50 Years Old With Myocarditis.

Background Myocarditis is an important cause of acute and chronic heart failure. Men with myocarditis have worse recovery and an increased need for transplantation compared with women, but the reason for the sex difference remains unclear. Elevated sera soluble (s) ST2 predicts mortality from acute and chronic heart failure, but has not been studied in myocarditis patients. Methods and Results Adults with a diagnosis of clinically suspected myocarditis (n=303, 78% male) were identified according to the 2013 European Society of Cardiology position statement. Sera sST2 levels were examined by ELISA in humans and mice and correlated with heart function according to sex and age. Sera sST2 levels were higher in healthy men ( P=8×10-6) and men with myocarditis ( P=0.004) compared with women. sST2 levels were elevated in patients with myocarditis and New York Heart Association class III - IV heart failure ( P=0.002), predominantly in men ( P=0.0003). Sera sST2 levels were associated with New York Heart Association class in men with myocarditis who were ≤50 years old ( r=0.231, P=0.0006), but not in women ( r=0.172, P=0.57). Sera sST2 levels were also significantly higher in male mice with myocarditis ( P=0.005) where levels were associated with cardiac inflammation. Gonadectomy with hormone replacement showed that testosterone ( P<0.001), but not estradiol ( P=0.32), increased sera sST2 levels in male mice with myocarditis. Conclusions We show in a well-characterized subset of heart failure patients with clinically suspected and biopsy-confirmed myocarditis that elevated sera sST2 is associated with an increased risk of heart failure based on New York Heart Association class in men ≤50 years old.

Genome-wide association study of cardiotoxicity in the NCCTG N9831 (Alliance) adjuvant trastuzumab trial.

OBJECTIVES: The major clinical side effect of the ERBB2-targeted breast cancer therapy, trastuzumab, is a decline in the left ventricular ejection fraction (LVEF). Improved markers are needed to better identify patients susceptible to cardiotoxicity. METHODS: The NCCTG N9831 trial compared adjuvant doxorubicin and cyclophosphamide followed by either weekly paclitaxel (arm A); paclitaxel then trastuzumab (arm B); or concurrent paclitaxel and trastuzumab (arm C) in patients with HER2-positive breast cancer. A genome-wide association study was performed on all patients with available DNA (N=1446). We used linear regression to identify single nucleotide polymorphisms (SNPs) associated with decline in LVEF, adjusting for age, baseline LVEF, antihypertensive medications, and the first two principle components. RESULTS: In total, 618 863 SNPs passed quality control and DNA from 1191 patients passed genotyping quality control and were identified as Whites of non-Hispanic origin. SNPs at six loci were associated with a decline in LVEF (P=7.73×10 to 8.93×10), LDB2, BRINP1, chr6 intergenic, RAB22A, TRPC6, and LINC01060, in patients who received chemotherapy plus trastuzumab (arms BC, N=800). None of these loci were significant in patients who received chemotherapy only (arm A, N=391) and did not increase in significance in the combined analysis of all patients. We did not observe association, P<0.05, with SNPs previously associated with trastuzumab-induced cardiotoxicity at ERBB2, I655V, and P1170A. We replicated association, P<0.05, with SNPs previously associated with anthracycline-induced cardiotoxicity at CBR3 and ABCB1. CONCLUSION: Our study identified six putative novel cardiotoxicity loci in patients treated with combination chemotherapy and trastuzumab that require further investigation and confirmed known associations of anthracycline-induced cardiotoxicity.