Ӧzcan, Ilke

Abstract Endothelial dysfunction can be regarded as the earliest feature of vascular aging and may occur in individuals without traditional risk factors for cardiovascular disease. Once present, endothelial dysfunction is known to increase the risk for atherosclerosis, cardiovascular diseases and complications such as stroke or death. Identification of individuals with early features of vascular aging remains challenging and has fuelled investigative efforts to associated biologic, imaging, and digital markers with endothelial dysfunction. This review highlights emerging and traditional methods to identify patients with early endothelial dysfunction and summarizes available and experimental therapies aimed at mitigating the vascular aging process.

Background: Cardiac allograft vasculopathy (CAV) is one of the leading causes of graft failure and death after heart transplantation (HTx). Conventionally, CAV is characterized by concentric intimal thickening, however the long-term progression patterns of plaque morphology are not yet well understood. Research Question: This study aimed to assess the plaque morphology, including histology and eccentricity, in a temporal manner post-HTx using virtual histology intravascular ultrasonography (VH-IVUS). Methods: We included 50 patients who underwent serial VH-IVUS assessment at three assessment points (A1:0-1 year, A2:3-5 years, A3:7-10 years) after HTx. Differences in VH-IVUS findings between timepoints A1 to A2 (early period) and A2 to A3 (late period) were investigated. Plaque volume index was defined as (plaque volume/vessel volume) × 100%. Eccentricity index (EI) was defined as (maximum intimal thickness – minimum intimal thickness)/maximum intimal thickness and was evaluated in 39 patients with significant plaque (maximal intimal thickness >0.50 mm at the A3) at each timepoint. The serial changes of EI and relationship between EI and other VH-IVUS findings were investigated. Results: Of 50 patients, mean age was 51±13 years, 36 (72%) were males. In both early and late periods, the plaque volume (PV) index showed a significant increase, however, the rate of progression was significantly slower in late period. (5.8% [0.9, 13.2] vs 2.4% [-0.2, 7.4], p=0.015). The EI showed a trend of decrease in the early period, compared to increases in the late period (-0.040 [-0.103, 0.004] vs 0.003 [-0.030, 0.052], p=0.036). Patients with EI change in late period higher than median value had significantly lower proportion of fibrous plaque (70.8% [67.2, 75.1] vs 62.7 [55.5, 66.9], p=0.002) and higher dense calcium (1.1% [0.9, 1.9], 4.5% [1.8, 6.9], p=0.022) at A3. Conclusion: In heart transplant patients, coronary artery plaque progression in the late period showed more eccentric progression with lower fibrous plaque composition, a pattern commonly observed in typical and conventional coronary atherosclerosis. These findings may provide a novel mechanism and therapeutic insight into the temporal patterns in CAV progression during long term follow up.

Abstract Background Cardiovascular disease remains the leading cause of death and burden globally, warranting the need to explore novel predictors of major adverse cardiovascular events (MACE) using new technologies. Artificial Intelligence (AI)-enhanced ECG (AI-ECG) algorithms can provide information on cardiovascular health of individuals, independent of conventional risk factors. Purpose The current study aimed to understand whether a previously described AI-ECG sex estimation algorithm could be applied for risk stratification of major adverse cardiovascular events (MACE), with a focus on a population without previous history of MACE. Methods Patients having 12-lead ECGs between 2018-2019 were included and followed up until August 2023. Exclusion criterion was a prior history of MACE. AI-ECG sex estimation was determined using a previously described algorithm predicting sex based on 12-lead ECGs. The algorithm generates outputs ranging from 0 to 1, representing the estimated probability of being male. Outputs above 0.5 were considered as AI-ECG-estimated male, and below as AI-ECG-estimated female. This was compared with biological sex to determine whether the two matched. Inconsistency between AI-ECG estimated sex and biological sex was defined as discordance. MACE was defined as myocardial infarction, coronary revascularization, non-fatal stroke, and all-cause death. Results Among 80,578 subjects (mean age 59.1 ± 16.1 years, 52.4% female), 9.7% exhibited sex discordance in AI-ECG estimation. Discordance was associated with a 56% higher risk of MACE (HR, 1.56, 95% CI 1.46-1.65, p <0.001), and remained significant in multivariable analysis with traditional risk factors (HR 1.35, 95% CI 1.27-1.43, p <0.001) (Figure 1). Sex-specific analysis showed increased MACE risk in both discordant males and females (Figure 2). Conclusion In patients with no history of MACE, discordance in AI-ECG sex estimation is associated with increased risk of MACE. These findings suggest the potential of AI-ECG sex estimation algorithm for risk assessment, especially in patients with no history of MACE.Figure 1.Cox proportional hazard modelFigure 2.Kaplan-Meier of MACE by sex

Abstract Background Vascular aging is a key feature of cardiovascular disease. α-klotho (klotho) is a protein inversely associated with aging and may contribute to vascular repair. Lower levels of systemic klotho have been identified in humans with features of early and advanced coronary atherosclerosis. It remains unknown if circulating klotho is present within human coronary circulation and whether cardiovascular klotho retention correlates with atherosclerosis. Purpose The current study aimed to assess the presence of klotho within coronary circulation compared to systemic circulation and hypothesized that net coronary klotho retention is attenuated in patients with early age-related features of coronary artery disease (CAD). Methods A total of 42 patients that underwent clinically indicated coronary angiogram for chest pain that had no obstructive CAD underwent intravascular ultrasound (IVUS) assessment were retrospectively examined. Virtual histology assessed coronary plaque characteristics. Blood samples simultaneously collected from the proximal aorta and coronary sinus were used for klotho detection using enzyme-linked immunosorbent assay. The net gradient of klotho across cardiovascular circulation was calculated by multiplying the arteriovenous klotho gradient by basal coronary blood flow. A net positive gradient indicated less cardiovascular klotho retention; a net negative gradient indicated greater cardiovascular klotho retention. Results Overall, participants were age 54±10 years old and 76% were female. There were overall higher levels of klotho within the coronary sinus (median 0.49 ng/mL [0.15; 1.69]) compared to systemic circulation (median 0.30 ng/mL [0.16; 1.14]). Participant groups were determined by those with positive (n = 20) or negative (n = 22) cardiovascular retention of klotho. Both groups were similar in age (56 ±2 vs. 51 ±2 years old, p = 0.080), gender (72% vs. 80% female, p = 0.580), and had similar occurrences of traditional cardiovascular comorbidities. Those with less cardiovascular klotho retention in the coronary vascular bed had significantly greater degree of non-obstructive coronary artery plaque (Gensini score 3.5 vs. 0, p < 0.001) and had higher percent plaque burden (46% [35; 53] vs. 34% [28; 46], p = 0.044) than those with greater cardiovascular klotho retention. Conclusions Patients with early coronary atherosclerosis may be characterized by attenuated retention of klotho within coronary circulation. Moreover, humans appear to have higher circulating klotho in coronary circulation than in systemic circulation. The current study may support a role for mechanism or potential therapeutic target for klotho in coronary atherosclerosis in humans.

Background Myocardial bridging (MB) is accompanied by the dynamic extravascular compression of epicardial coronary arteries, leading to intracoronary hemodynamic disturbance with abnormal coronary flow profiles. We aimed to evaluate the prognostic implications of resistive reserve ratio (RRR), a composite measure of flow and pressure parameters that represents the vasodilatory capacity of the coronary arteries, in patients with angina with nonobstructive coronary artery disease (ANOCA) and MB, in comparison with coronary flow reserve (CFR). Methods and Results In this retrospective cohort study, we included patients with ANOCA who underwent coronary reactivity testing, where MB was identified by transient constriction in coronary artery segments between systole and diastole. Abnormal CFR and RRR were defined as <2.5 and <2.62, respectively. Major adverse cardiac events, including cardiovascular death, stroke, myocardial infarction, heart failure, and late revascularization, served as outcomes. Among 1251 patients with ANOCA, 191 (15.3%) had MB. The prevalence of abnormal CFR or RRR was not significantly different between patients with and without MB ( P =0.144 and P =0.398, respectively). Over a median follow‐up time of 6.9 years, abnormal RRR predicted major adverse cardiac events in patients with MB (hazard ratio [HR], 4.38 [95% CI, 1.71–11.21]; P =0.002) and without MB (HR, 1.91 [95% CI, 1.38–2.64]; P <0.001). Abnormal CFR predicted major adverse cardiac events in patients without MB (HR, 2.15 [95% CI, 1.54–3.00]; P <0.001), whereas it was not predictive of major adverse cardiac events in patients with MB (HR, 2.29 [95% CI, 0.93–5.65]; P =0.073). Conclusions In patients with ANOCA and MB, impaired RRR was superior to impaired CFR in distinguishing patients at a higher risk of future adverse events, suggesting that RRR may serve as a risk stratification tool in patients with MB and ANOCA.

Introduction: The endothelium controls coronary blood flow (CBF) by regulating microvascular tone, which is an important index of microcirculatory health. The difference in vasomotor response of the microvasculature between males and females is not well described. Hypothesis: Males and females react differently to different doses of intracoronary (IC) acetylcholine (Ach) injections. Methods: Patients with angina and non-obstructive coronary artery disease (<50% stenosis) that underwent coronary reactivity study were enrolled. A doppler wire was positioned and incremental IC Ach doses were injected (10 -6 , 10 -5 , and 10 -4 mol/L). CBF (mL/min) was then calculated from CAD and doppler-derived flow velocity. Comparisons between the 2 sexes were performed. Results: A total of 1927 patients (mean age 51±13 years, 67% females) were included. Males were significantly younger (49±13 vs 53±12, P<0.0001) than females and had a higher smoking exposure rate (56% vs 41%, P<0.0001). Baseline (B) CBF was significantly lower in males (27.7 [19.2; 40.8] vs 30.2 [20.6; 43.4], P=0.04). The gap between females and males widened the first 2 doses, but then diminished after the highest dose (41.2 [24.5; 67.8] vs 44.5 [25.9; 72.0], P=0.09) (Fig 1A) . On multivariable analyses, female sex was an independent predictor of higher CBF at all doses (Estimate±StdError) (B= 1.14±0.53; 10 -6 : 1.59±0.61; 10 -5 : 2.54±0.82; 10 -4 : 2.70±1.2; P<0.05 for all). When calculating the %ΔCBF, females had a significantly larger augmentation of flow after the first 2 doses of IC Ach (10 -6 : 3 [-14; 24] vs. -2 [-16; 19], P=0.02; 10 -5 : 21 [-7;68] vs. 12 [-10; 50], P=0.003). However, with the highest Ach dose, there was an equalization of overall augmentation (10 -4 : 49 [-1; 109] vs. 46 [-5; 106], P=0.46) (Fig 1B). Conclusions: Females had higher CBF regardless of given Ach concentration. Females have a higher sensitivity to Ach at smaller doses, while males exhibit a greater vasodilatory reaction at higher doses.

Introduction: Coronary microvascular dysfunction (CMD) is associated with increased morbidity and mortality. Anemia is an important predictor of outcomes in several cardiovascular diseases, such as heart failure and coronary artery disease (CAD), however its significance in patients with non-obstructive CAD is not fully elucidated. Hypothesis: We hypothesized that anemia would be associated with CMD and future adverse outcomes. Methods: We enrolled 1334 patients with signs and/or symptoms of ischemia and with non-obstructive CAD (<50% angiographic stenosis) who underwent invasive CMD evaluation and had follow-up data. CMD was defined as coronary flow reserve (CFR) (hyperemic flow/baseline flow as measured by the doppler wire) of ≤2 in response to intracoronary adenosine injection. Anemia was defined as a Hgb < 13.0 g/L in males and <12.0 g/L in females. MACE was defined as any incidence of myocardial infarction, cardiac revascularization (PCI, CABG) and all-cause mortality. Results: Overall mean age was 51.3±12.4 years, 66% were female. Patients with anemia were more likely to be female and have diabetes mellitus. Anemics were more likely to be diagnosed with CMD (Odds Ratio 2.01, 95% Confidence Interval [1.14-3.54]; P = 0.02). This relationship remained significant even after adjusting for cardiovascular risk factors like age, sex, hypertension, diabetes mellitus and dyslipidemia (OR 1.84, 95% CI [1.03-3.29], P = 0.04). Furthermore, patients with anemia were more likely to experience MACE (OR 1.86, 95% CI [1.30-2.66], P = 0.001), independent of possible confounders (OR 1.84, 95% CI [1.27-2.69], P = 0.002). Conclusions: Anemia is an independent predictor of CMD and future adverse cardiovascular events in patients with NOCAD. Moreover, it was associated with an increased risk of future adverse events. Further studies are needed to elucidate whether treatment of anemia in this population translates into outcomes.

Introduction: Changes in coronary artery diameter (CAD) and coronary blood flow (CBF) in reaction to acetylcholine are important surrogates of endothelial health, and abnormalities in these parameters might represent early atherosclerosis. Menopause is associated with an increased risk of coronary artery disease, yet the role of menopause on the endothelium is not well described. Hypothesis: The vasodilatory response of the endothelium to acetylcholine (Ach) decreases after menopause. Methods: A total of 1271 (42% pre-menopausal) females with angina and non-obstructive coronary artery disease (<50% angiographic stenosis) that underwent coronary reactivity study were enrolled. A doppler wire was positioned and incremental Ach doses were injected (10 -6 , 10 -5 , and 10 -4 mol/L). CBF was then calculated from CAD and doppler-derived flow velocity. Comparisons between the pre-and post-menopause groups were performed. Results: Mean age of the pre-menopausal group was 44±10 years, while that of the post-menopausal 59±9 years. Pre-menopausal females had higher baseline CAD as compared to post-menopausal females (2.22 [1.90; 2.65] vs. 2.20 [1.85; 2.55] mm, P=0.03). However, this difference was nullified with subsequent doses of Ach (Figure 1A&B ). Pre-menopausal females had a higher baseline CBF than post-menopausal females (32.1 [21.7; 44.9] vs. 29.2 [19.8; 42.0] mL/min; P=0.01). This difference progressively increased with successive doses of Ach (10 -4 :49.5 [30.3; 77.0] vs. 41.7 [24.4; 69.0] mL/min; P=0.0002), revealing a greater microvascular vasodilatory response in pre-menopausal females (Cumulative %ΔCBF after highest dose= 60 [2.6; 122] vs 45 [-4; 98], P=0.002) ( Figure 1C&D ). Conclusions: Premenopausal females have greater sensitivity of the microvascular endothelium to Ach compared to post-menopausal, suggesting a possible role for estrogen in mediating the effect of stress on the microvasculature through the endothelium.

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.

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.