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Introduction: Diabetes mellitus and insulin resistance are associated with accelerated vascular injury and progression of coronary atherosclerosis. CD34 + progenitor cells are mobilized and recruited to repair the endothelial and vascular damage. Hypothesis: We hypothesized that the interaction of insulin levels and retention of CD34+ progenitors in the coronary arteries will be different between patients with and without insulin resistance. Methods: In patients with no obstructive coronary artery disease (NOCAD: <40% angiographic stenosis), blood samples were collected concurrently from the proximal aorta and the coronary sinus, and CD34+ cells counted using flow cytometry. CD34+ cells gradient was calculated by subtracting aortic from coronary sinus cell counts, with a higher gradient reflecting lower retention. Patients on antidiabetic medications were excluded. Insulin resistance (IR) was estimated as HOMA-IR index ([insulin]*[glucose]/405)) ≥2. Results: Of 128 patients (mean age 51±11 years, 77% females), 60 (47%) had IR (mean age 51±10 years, 73% females) and 68 (53%) did not (mean age 50±13 years, 79% females). The groups did not differ in age, sex or LDL cholesterol. The IR group had higher BMI and triglyceride levels and lower HDL cholesterol. CD34 + cell gradient correlated directly significantly with insulin levels in patients without IR (r=0.25, p=0.04) (Fig1A) , but inversely in those with IR (r=-0.39, p=0.01) (Fig1B) . Both associations remained significant after adjustment for age, sex, and BMI (IR: r=-0.39, p=0.02; no IR: r=29, p=0.04). Conclusions: In NOCAD patients with IR, higher levels of serum insulin were associated with increased retention of CD34 + cells in the coronary arteries. The current study supports a potential role for CD34 + cells for vascular repair in patients with IR.

Objective To test the hypothesis that the numbers of circulating endothelial progenitor cells (EPC) co-expressing an osteogenic phenotype (EPC-OC) are increased in patients with clinically severe calcific aortic valve stenosis (AoC). Background AoC is one of the most common valvular heart diseases. Recent studies have shifted the old paradigm of AoC being a passive, degenerative process to an active biological process associated with an osteoblast-like phenotype. We have recently identified circulating EPC-OC which are significantly increased in patients with severe coronary artery disease (CAD). Methods and Results Of 38 patients, 14 underwent aortic valve replacement due to AoC with only mild angiographic CAD. Patients with normal aortic valves and coronary arteries (n=12) or severe CAD without aortic valve disease (n=12) served as controls (Table ). Peripheral blood mononuclear cells were analyzed using flow cytometry following staining for EPC markers (CD133, CD34, KDR) and the osteoblastic marker, osteocalcin (OC). Patients with AoC had significantly higher fractions of OC co-expression on EPC of different maturity than normal patients, similar to patients with CAD (Figure ). Conclusions Circulating EPC co-expressing an osteoblastic phenotype are increased and may potentially play a role in the active process of aortic valve calcification. Table. Clinical Data.

Background Coronary microvascular dysfunction (CMD) represents an early functional characteristic of coronary vascular aging. Klotho (α‐klotho) is a circulating protein inversely linked to physiological aging. We examined low klotho as a potential marker for vascular aging in patients with CMD and no coronary artery disease. Methods and Results Patients undergoing nonurgent angiogram for chest pain who had no coronary artery disease underwent invasive coronary microvascular and endothelial function testing. CMD was defined by ≤50% increase in coronary blood flow (percentage change in coronary blood flow) in response to intracoronary acetylcholine or coronary flow reserve ≤2. Fresh arterial whole blood was used to analyze circulating endothelial progenitor cells with flow cytometry. Stored arterial plasma was used for klotho analysis by ELISA. Participants with CMD (n=62) were compared with those without CMD (n=36). Those with CMD were age 55±10 years (versus 51±11 years; P =0.07) and 73% women (versus 81%; P =0.38). Traditional risk factors for coronary artery disease were similar between groups. Patients with CMD had less klotho (0.88±1.50 versus 1.75±2.38 ng/mL; P =0.03), and the odds of low klotho in CMD were significant in a logistic regression model after adjusting for traditional cardiovascular risk factors (odds ratio [OR], 0.80 [95% CI, 0.636–0.996]; P =0.05). Higher klotho was associated with higher numbers of endothelial progenitor cells with vascular regenerative potential (CD34 + and CD34 + CD133 + KDR + ). Among a subgroup of patients with atherosclerotic cardiovascular disease risk <5% (n=58), CMD remained associated with lower klotho (OR, 0.80 [95% CI, 0.636–0.996]; P =0.047). Conclusions Klotho may be a biomarker for CMD and may be a therapeutic target for groups of patients without significant traditional cardiovascular risk.

Endothelial cell (EC) injury is a crucial contributor to the progression of diabetic kidney disease (DKD), but the specific EC populations and mechanisms involved remain elusive. Kidney ECs (n = 5464) were collected at three timepoints from diabetic BTBRob/ob mice and non-diabetic littermates. Their heterogeneity, transcriptional changes, and alternative splicing during DKD progression were mapped using SmartSeq2 single-cell RNA sequencing (scRNAseq) and elucidated through pathway, network, and gene ontology enrichment analyses. We identified 13 distinct transcriptional EC phenotypes corresponding to different kidney vessel subtypes, confirmed through in situ hybridization and immunofluorescence. EC subtypes along nephrons displayed extensive zonation related to their functions. Differential gene expression analyses in peritubular and glomerular ECs in DKD underlined the regulation of DKD-relevant pathways including EIF2 signaling, oxidative phosphorylation, and IGF1 signaling. Importantly, this revealed the differential alteration of these pathways between the two EC subtypes and changes during disease progression. Furthermore, glomerular and peritubular ECs also displayed aberrant and dynamic alterations in alternative splicing (AS), which is strongly associated with DNA repair. Strikingly, genes displaying differential transcription or alternative splicing participate in divergent biological processes. Our study reveals the spatiotemporal regulation of gene transcription and AS linked to DKD progression, providing insight into pathomechanisms and clues to novel therapeutic targets for DKD treatment.

Background Recent studies have indicated high rates of future major adverse cardiovascular events in patients with Takotsubo cardiomyopathy (TC), but there is no well‐established tool for risk stratification. This study sought to evaluate the prognostic value of several artificial intelligence‐augmented ECG (AI‐ECG) algorithms in patients with TC. Methods and Results This study examined consecutive patients in the prospective and observational Mayo Clinic Takotsubo syndrome registry. Several previously validated AI‐ECG algorithms were used for the estimation of ECG‐ age, probability of low ejection fraction, and probability of atrial fibrillation. Multivariable models were constructed to evaluate the association of AI‐ECG and other clinical characteristics with major adverse cardiac events, defined as cardiovascular death, recurrence of TC, nonfatal myocardial infarction, hospitalization for congestive heart failure, and stroke. In the final analysis, 305 patients with TC were studied over a median follow‐up of 4.8 years. Patients with future major adverse cardiac events were more likely to be older, have a history of hypertension, congestive heart failure, worse renal function, as well as high‐risk AI‐ECG findings compared with those without. Multivariable Cox proportional hazards analysis indicated that the presence of 2 or 3 high‐risk findings detected by AI‐ECG remained a significant predictor of major adverse cardiac events in patients with TC after adjustment by conventional risk factors (hazard ratio, 4.419 [95% CI, 1.833–10.66], P =0.001). Conclusions The combined use of AI‐ECG algorithms derived from a single 12‐lead ECG might detect subtle underlying patterns associated with worse outcomes in patients with TC. This approach might be beneficial for stratifying high‐risk patients with TC.

Introduction: α-Klotho (klotho) is a geroprotective protein inversely associated with coronary artery disease CAD and vascular aging. This study used intravascular ultrasound (IVUS) to study coronary artery plaque characteristics of patients with low circulating klotho. Hypothesis: Lower klotho is associated with vulnerable coronary artery plaque characteristics. Methods: Patients undergoing coronary angiogram for chest pain who had no CAD (<40% stenosis) underwent IVUS assessment. Images were obtained from the mid-LAD to the coronary ostium. Stored arterial plasma was used for α-klotho analysis by enzyme-linked immunosorbent assay. Results: A total of 58 consecutive patients were retrospectively studied. The median circulating klotho level of the cohort was 0.27 ng/mL [0.15; 1.24]. Patients with klotho less than the median were compared to those with levels greater than or equal to the median and were similar in age (53 ±11 vs. 56 ±10, p = 0.235) and sex distribution (86% vs. 76% female, p = 0.324). Patients with less klotho had higher coronary plaque burden (21.42% [17.185, 31.955] vs. 18.32 [10.5, 24.515], p=0.041) and larger plaque area (2.71 [2.03, 4.605] vs . 2.27 [1.41, 3.31] mm 3 /mm, p=0.058) compared to those with higher klotho. Lower klotho was associated with vulnerable plaque characteristics, including greater percentages of dense calcium (1.33% [0.265, 5.38] vs. 0.43% [0.015, 1.73], p=0.037) and necrotic core (5.43% [2.245, 10.5] vs. 2.22% [0.86, 4.50], p=0.011). Conclusions: Lower circulating klotho was associated with higher coronary plaque burden and vulnerable plaque characteristics. Klotho may be an early biomarker for risk in coronary artery disease.

The single-cell platform mass cytometry (CyTOF) enables detection of significant changes in one million of renal cells, especially in subsets of immune cells in ischemic human kidneys distal to renal artery stenosis (RAS). We found that pro-repair cell types such as scattered tubular-progenitor-like cells, aged neutrophils, and regulatory B cells show a compensatory increase in RAS. Immune cell phenotype changes may reflect ongoing inflammation and impaired immune defense capability in the kidneys.

Monoamine Oxidase (MAO), an outer mitochondrial enzyme, catalyzes the oxidative deamination of neurotransmitters and biological amines, and in the process, produces a reactive aldehyde and H2O2. Our lab has recently shown that MAO activity (i.e., H2O2 production) in human right atrial myocardium is associated with postoperative atrial fibrillation. Furthermore, our lab has shown that diabetic patients have higher MAO‐A and ‐B content and rates of activity in this tissue, compared to nondiabetics. While recent studies have explored the connection between MAO and cardiac dysfunction, the chamber‐specific content and activity of MAO within the heart is still unknown. Therefore, we characterized the regional enzymatic activity of MAO‐A and ‐B based upon the hypothesis that the atria would have the highest MAO activity due to the greater density of local sympathetic nerve innervation in this region. For this study, porcine hearts were isolated from domestic pigs, as this model closely recapitulates the physiology of the human heart. One group of pigs were fed a high fat, high sucrose diet for 16 weeks to induce obesity/metabolic syndrome (n=4). Three MAO substrates (dopamine, norepinephrine, serotonin) and two isoform‐selective inhibitors (clorgyline=MAO‐A, deprenyl=MAO‐B) were used to characterize MAO activities. Enzyme content was measured using ELISA and confirmed with immunoblots. Kinetic analysis showed that, across all substrates, when normalized to tissue protein, the left atrium has the highest maximal MAO‐A and –B activity while the other three chambers are similar. Furthermore, maximal MAO activity in the left ventricle of pigs with metabolic syndrome is significantly greater (P<0.05) than left ventricle of control group. Our findings suggest that MAO activity differs by location in the heart (i.e., chamber heterogeneity). This information will be useful to inform future experimental and clinical pharmacology applications where MAO inhibitors might be used. Moreover, increased MAO in left ventricle of pigs with obesity/metabolic syndrome is important as it provides a rationale for exploring MAO inhibitors as therapeutic strategy to mitigate cardiac complications of diabetes. Support or Funding Information R01HL122863