Ubiquitination is a representative, reversible biological process of the post-translational modification of various proteins with multiple catalytic reaction sequences, including ubiquitin itself, in addition to E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, E3 ubiquitin ligase, deubiquitinating enzymes, and proteasome degradation. The ubiquitin–proteasome system is known to play a pivotal role in various molecular life phenomena, including the cell cycle, protein quality, and cell surface expressions of ion-transporters. As such, the failure of this system can lead to cancer, neurodegenerative diseases, cardiovascular diseases, and hypertension. This review article discusses Nedd4-2/NEDD4L, an E3-ubiquitin ligase involved in salt-sensitive hypertension, drawing from detailed genetic dissection analysis and the development of genetically engineered mice model. Based on our analyses, targeting therapeutic regulations of ubiquitination in the fields of cardio-vascular medicine might be a promising strategy in future. Although the clinical applications of this strategy are limited, compared to those of kinase systems, many compounds with a high pharmacological activity were identified at the basic research level. Therefore, future development could be expected.

The epithelial sodium channel (ENaC) plays critical roles in maintaining fluid and electrolyte homeostasis and is located in the aldosterone-sensitive distal nephron (ASDN). We previously found that Nedd4-2 C2 knockout (KO) mice showed salt-sensitive hypertension with paradoxically enhanced ENaC gene expression in ASDN under high oral salt intake. Eplerenone (EPL), a selective aldosterone blocker, is a promising therapeutic option for resistant or/and salt-sensitive hypertension. We examined the effect of EPL on Nedd4-2 C2 KO mice with respect to blood pressure, metabolic parameters, and molecular level changes in ASDN under high oral salt intake. We found that EPL failed to reduce blood pressure in KO mice with high oral salt intake and upregulated ENaC expression in ASDN. Thus, salt-sensitive hypertension in Nedd4-2 C2 KO was EPL-resistant. Gene expression analyses of laser-captured specimens in ASDN suggested the presence of non-aldosterone-dependent activation of ENaC transcription in ASDN of Nedd4-2 C2 KO mice, which was abolished by amiloride treatment. Our results from Nedd4-2 C2 KO mice suggest that enhanced ENaC gene expression is critically involved in salt-sensitive hypertension under certain conditions of specific enzyme isoforms for their ubiquitination.

The prorenin/renin receptor ((P)RR) is a multifunctional protein that is widely distributed in various organs. Despite intensive research for more than 20 years, this receptor has not been fully characterized. In this study, we generated mice overexpressing the tubular epithelial (P)RR gene ((P)RR-TG mice) to test the previously reported functional role of (P)RR by Ramkumar et al. in 2015 using tubular specific (P)RR KO mice. (P)RR-TG mice were maintained and analyzed in individual metabolic cages and were administered angiotensin II blocker (ARB), direct renin inhibitor (DRI), and bafilomycin, that is, vacuolar ATPase (V-ATPase) antagonist. (P)RR-TG mice were hypertensive and had alkalized urine with lower osmolality and Na⁺ excretion. ARB and DRI, but not bafilomycin, concurrently decreased blood pressure. Bafilomycin acidized urine of (P)RR-TG mice, or equivalently this phenomenon restored the effect of overexpressed transgene, suggesting that (P)RR functioned as a V-ATPase in renal tubules. Afterall, (P)RR-TG mice were mated with alternative renin transgenic mice (ARen2-TG), which we identified as intracellular renin previously, to generate double transgenic mice (DT-TG). Lethal renal tubular damage was observed in DT-TG mice, suggesting that intracellular renin may be a ligand for (P)RR in tubules. In summary, (P)RR did not substantially affect the tissue renin-angiotensin system (RAS) in our model of tubular specific (P)RR gene over-expression, but alternative intracellular renin may be involved in (P)RR signaling in addition to conventional V-ATPase function. Further investigations are warranted.

Background: Cardiovascular diseases (CVDs) remain a leading cause of morbidity and mortality, despite advances in treatment. Early detection of vascular aging is critical, as preclinical atherosclerosis often remains undiagnosed. AI-determined vascular age, originally developed using carotid-femoral pulse wave velocity (cf-PWV), may help to identify individuals at elevated risk. This study aimed to evaluate the clinical utility of an alternative AI-determined vascular age model based on the arterial velocity pulse index (AVI) and arterial pressure volume index (API) in a Japanese hospital-based cohort. Methods: This retrospective, exploratory study analyzed electronic health records of 408 patients from Yokohama City University Hospital. This study was approved by the Clinical Research Ethics Committee (approval numbers: B180300040, F240500007), and patient consent was obtained through an opt-out process. AI-determined vascular age was estimated using a Generalized Additive Model (GAM) with backward stepwise regression, substituting cf-PWV with AVI and API. Correlations with chronological age were assessed, and comparisons of cardiovascular and renal function markers were performed across age-stratified groups. Results: AI-determined vascular age showed a strong correlation with chronological age (p < 0.05). Significant differences were observed in cardiac diastolic function parameters, B-type natriuretic peptide (BNP), and estimated glomerular filtration rate (eGFR) between the highest and lowest quintiles of AI-determined vascular age. Conclusions: AI-determined vascular age using AVI and API appears to be a feasible surrogate for cf-PWV in clinical settings. This index may aid in stratifying vascular aging and identifying individuals who could benefit from early cardiovascular risk management.

Background: The Arterial Velocity Pulse Index (AVI) and Arterial Pressure–Volume Index (API) are novel non-invasive indices of arterial stiffness derived from cuff-oscillometric measurements. Previous studies have shown that elevated AVI and API are associated with the severity of coronary artery disease and the ability to predict future cardiovascular events. However, the hemodynamic and echocardiographic characteristics of patients with concomitantly high AVI and API remain unclear. Methods: We retrospectively analyzed 112 consecutive cardiovascular outpatients (mean age 69.1 ± 12.2 years, 64.3% male) seen between January and April 2019 at Yokohama City University Hospital. The AVI and API were measured using a multifunctional sphygmomanometer (PASESA AVE1500, Shisei Datum, Japan) and averaged over a maximum of three measurements. Patients were classified into four groups according to previously established cutoff values (AVI ≥ 27, API ≥ 32). Central arterial pulse wave parameters were assessed using SphygmoCor XCEL (AtCor Medical, Sydney, Australia), and echocardiographic parameters were obtained according to standard protocols. Intergroup differences were analyzed using the Kruskal–Wallis test with Steel–Dwass post hoc comparisons. Results: Compared with the low-risk group (low AVI/low API), the high-risk group (high AVI/high API) had significantly higher brachial systolic BP (139.2 [132.8–149] vs. 128 [120–136.7] mmHg, p = 0.0011), central systolic BP (127.5 [122.3–139] vs. 117.7 [110.3–123.7] mmHg, p = 0.0018), and central pulse pressure (56.2 [51.4–60.3] vs. 37.7 [32–43] mmHg, p < 0.001). The forward and reflected wave amplitudes were significantly greater, with prolonged ejection duration and aortic T2 time. The Buckberg subendocardial viability ratio was significantly lower in the high-risk group (129.5 [119.7–145.2] vs. 148.3 [130–168.3], p = 0.040). Echocardiography revealed reduced e′ velocity (5 [4.1–5.8] vs. 6.7 [5.2–8] cm/s, p = 0.035) and increased E/e′ (13.2 [11.1–15.1] vs. 9.7 [7.9–11.3], p = 0.026) in the high-risk group, suggesting the presence of impaired diastolic function without reduced LVEF. Conclusions: Patients with high AVI and API exhibited greater central and peripheral arterial stiffness, higher systolic and pulse pressures, and impaired diastolic function compared with those with low values. These findings support the use of a cardiovascular pathophysiological model in which elevated AVI/API identify individuals at increased risk of progression to heart failure and ischemic heart disease.

The neonatal heart possesses a unique capacity for reparative healing after myocardial injury, unlike the adult heart. While immune cells, particularly T cells, regulate post-infarction inflammation, their role in age-dependent cardiac repair remains unclear. This study aimed to characterize the temporal activation of T cell subsets and their contribution to immune homeostasis and myocardial repair. Myocardial infarction was induced in mice of different ages, and T cell subsets (CD4+ T cells, CD8+ T cells, and CD4+Foxp3+ T [T-reg] cells) were analyzed using flow cytometry and RNA sequencing. Neonatal hearts exhibited CD4+ T cells, CD8+ T cells, and T-reg cells that gradually increased until seven days post-injury. Transcriptome analysis identified Rcn3 as a neonatal-specific, injury-responsive gene in T-reg cells, with minimal induction in adult and aged hearts, promoting a reparative microenvironment and exerting anti-fibrotic effects via the PI3K/Akt pathway. Under endoplasmic reticulum stress, Rcn3 activated unfolded protein response genes, and Rcn3-conditioned media reduced fibrosis-associated gene expression in adult cardiac fibroblasts. In a conditional knockout mouse model (Lck-cre; Rcn3^fl/fl), Rcn3 deletion in T cells led to impaired cardiac function recovery and increased fibrosis post-injury. These findings suggest that neonatal T-reg cells play a crucial role in cardiac repair, with Rcn3 as a potential therapeutic target for enhancing immune-mediated cardiac repair and limiting pathological remodeling in the adult heart.