Advances in 3D Organoid Models for Stem Cell-Based Cardiac Regeneration.

The adult human heart cannot regain complete cardiac function following tissue injury, making cardiac regeneration a current clinical unmet need. There are a number of clinical procedures aimed at reducing ischemic damage following injury; however, it has not yet been possible to stimulate adult cardiomyocytes to recover and proliferate. The emergence of pluripotent stem cell technologies and 3D culture systems has revolutionized the field. Specifically, 3D culture systems have enhanced precision medicine through obtaining a more accurate human microenvironmental condition to model disease and/or drug interactions in vitro. In this study, we cover current advances and limitations in stem cell-based cardiac regenerative medicine. Specifically, we discuss the clinical implementation and limitations of stem cell-based technologies and ongoing clinical trials. We then address the advent of 3D culture systems to produce cardiac organoids that may better represent the human heart microenvironment for disease modeling and genetic screening. Finally, we delve into the insights gained from cardiac organoids in relation to cardiac regeneration and further discuss the implications for clinical translation.

Shear stress regulation of miR-93 and miR-484 maturation through nucleolin.

Pulsatile shear (PS) and oscillatory shear (OS) elicit distinct mechanotransduction signals that maintain endothelial homeostasis or induce endothelial dysfunction, respectively. A subset of microRNAs (miRs) in vascular endothelial cells (ECs) are differentially regulated by PS and OS, but the regulation of the miR processing and its implications in EC biology by shear stress are poorly understood. From a systematic in silico analysis for RNA binding proteins that regulate miR processing, we found that nucleolin (NCL) is a major regulator of miR processing in response to OS and essential for the maturation of miR-93 and miR-484 that target mRNAs encoding Krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS). Additionally, anti-miR-93 and anti-miR-484 restore KLF2 and eNOS expression and NO bioavailability in ECs under OS. Analysis of posttranslational modifications of NCL identified that serine 328 (S328) phosphorylation by AMP-activated protein kinase (AMPK) was a major PS-activated event. AMPK phosphorylation of NCL sequesters it in the nucleus, thereby inhibiting miR-93 and miR-484 processing and their subsequent targeting of KLF2 and eNOS mRNA. Elevated levels of miR-93 and miR-484 were found in sera collected from individuals afflicted with coronary artery disease in two cohorts. These findings provide translational relevance of the AMPK-NCL-miR-93/miR-484 axis in miRNA processing in EC health and coronary artery disease.

Atheroprotective Flow Upregulates ITPR3 (Inositol 1,4,5-Trisphosphate Receptor 3) in Vascular Endothelium via KLF4 (Krüppel-Like Factor 4)-Mediated Histone Modifications.

Objective- The topographical distribution of atherosclerosis in vasculature underscores the importance of shear stress in regulating endothelium. With a systems approach integrating sequencing data, the current study aims to explore the link between shear stress-regulated master transcription factor and its regulation of endothelial cell (EC) function via epigenetic modifications. Approach and Results- H3K27ac (acetylation of histone 3 lysine 27)-ChIP-seq (chromatin immunoprecipitation followed by high throughput sequencing), ATAC-seq (an assay for transposase-accessible chromatin-sequencing), and RNA-seq (RNA-sequencing) were performed to investigate the genome-wide epigenetic regulations in ECs in response to atheroprotective pulsatile shear stress (PS). In silico prediction revealed that KLF4 binding motifs were enriched in the PS-enhanced H3K27ac regions. By integrating PS- and KLF4-modulated H3K27ac, we identified 18 novel PS-upregulated genes. The promoter regions of these genes showed an overlap between the KLF4-enhanced assay for transposase-accessible chromatin signals and the PS-induced H3K27ac peaks. Experiments using ECs isolated from mouse aorta, lung ECs from EC-KLF4-TG versus EC-KLF4-KO mice, and atorvastatin-treated ECs showed that ITPR3 (inositol 1,4,5-trisphosphate receptor 3) was robustly activated by KLF4 and statins. KLF4 ATAC-qPCR (quantitative polymerase chain reaction) and ChIP-qPCR further demonstrated that a specific locus in the promoter region of the ITPR3 gene was essential for KLF4 binding, H3K27ac enrichment, chromatin accessibility, RNA polymerase II recruitment, and ITPR3 transcriptional activation. Deletion of this KLF4 binding locus in ECs by using CRISPR-Cas9 resulted in blunted calcium influx, reduced expression of endothelial nitric oxide synthase, and diminished nitric oxide bioavailability. Conclusions- These results from a novel multiomics study suggest that KLF4 is crucial for PS-modulated H3K27ac that allow the transcriptional activation of ITPR3. This novel mechanism contributes to the Ca2+-dependent eNOS (endothelial nitric oxide synthase) activation and EC homeostasis.

miR-483 Targeting of CTGF Suppresses Endothelial-to-Mesenchymal Transition: Therapeutic Implications in Kawasaki Disease.

RATIONALE: Endothelial-mesenchymal transition (EndoMT) is implicated in myofibroblast-like cell-mediated damage to the coronary arterial wall in acute Kawasaki disease (KD) patients, as evidenced by positive staining for connective tissue growth factor (CTGF) and EndoMT markers in KD autopsy tissues. However, little is known about the molecular basis of EndoMT involved in KD. OBJECTIVE: We investigated the microRNA (miRNA) regulation of CTGF and the consequent EndoMT in KD pathogenesis. As well, the modulation of this process by statin therapy was studied. METHODS AND RESULTS: Sera from healthy children and KD subjects were incubated with human umbilical vein endothelial cells. Cardiovascular disease-related miRNAs, CTGF, and EndoMT markers were quantified using reverse transcriptase quantitative polymerase chain reaction, ELISA, and Western blotting. Compared with healthy controls, human umbilical vein endothelial cell incubated with sera from acute KD patients had decreased miR-483, increased CTGF, and increased EndoMT markers. Bioinformatics analysis followed by functional validation demonstrated that Krüppel-like factor 4 (KLF4) transactivates miR-483, which in turn targets the 3' untranslated region of CTGF mRNA. Overexpression of KLF4 or pre-miR-483 suppressed, whereas knockdown of KLF4 or anti-miR-483 enhanced, CTGF expression in endothelial cells in vitro and in vivo. Furthermore, atorvastatin, currently being tested in a phase I/IIa clinical trial in KD children, induced KLF4-miR-483, which suppressed CTGF and EndoMT in endothelial cells. CONCLUSIONS: KD sera suppress the KLF4-miR-483 axis in endothelial cells, leading to increased expression of CTGF and induction of EndoMT. This detrimental process in the endothelium may contribute to coronary artery abnormalities in KD patients. Statin therapy may benefit acute KD patients, in part, through the restoration of KLF4-miR-483 expression. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01431105.

AMP-activated Protein Kinase Phosphorylation of Angiotensin-Converting Enzyme 2 in Endothelium Mitigates Pulmonary Hypertension.

RATIONALE: Endothelial dysfunction plays an integral role in pulmonary hypertension (PH). AMPK (AMP-activated protein kinase) and ACE2 (angiotensin-converting enzyme 2) are crucial in endothelial homeostasis. The mechanism by which AMPK regulates ACE2 in the pulmonary endothelium and its protective role in PH remain elusive. OBJECTIVES: We investigated the role of AMPK phosphorylation of ACE2 Ser680 in ACE2 stability and deciphered the functional consequences of this post-translational modification of ACE2 in endothelial homeostasis and PH. METHODS: Bioinformatics prediction, kinase assay, and antibody against phospho-ACE2 Ser680 (p-ACE2 S680) were used to investigate AMPK phosphorylation of ACE2 Ser680 in endothelial cells. Using CRISPR-Cas9 genomic editing, we created gain-of-function ACE2 S680D knock-in and loss-of-function ACE2 knockout (ACE2-/-) mouse lines to address the involvement of p-ACE2 S680 and ACE2 in PH. The AMPK-p-ACE2 S680 axis was also validated in lung tissue from humans with idiopathic pulmonary arterial hypertension. MEASUREMENTS AND MAIN RESULTS: Phosphorylation of ACE2 by AMPK enhanced the stability of ACE2, which increased Ang (angiotensin) 1-7 and endothelial nitric oxide synthase-derived NO bioavailability. ACE2 S680D knock-in mice were resistant to PH as compared with wild-type littermates. In contrast, ACE2-knockout mice exacerbated PH, a similar phenotype found in mice with endothelial cell-specific deletion of AMPKα2. Consistently, the concentrations of phosphorylated AMPK, p-ACE2 S680, and ACE2 were decreased in human lungs with idiopathic pulmonary arterial hypertension. CONCLUSIONS: Impaired phosphorylation of ACE2 Ser680 by AMPK in pulmonary endothelium leads to a labile ACE2 and hence is associated with the pathogenesis of PH. Thus, AMPK regulation of the vasoprotective ACE2 is a potential target for PH treatment.

TIFA as a crucial mediator for NLRP3 inflammasome.

Toll-like receptor-mediated NF-κB activation is a major innate immune reaction of vascular endothelial cells (ECs) in response to prooxidative and proinflammatory stimuli. We identified that TNF-α receptor-associated factor-interacting protein with a forkhead-associated domain (TIFA) is a regulator of priming (signal 1) and activating (signal 2) signals of nucleotide oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome in ECs. Oxidative and inflammatory stresses such as atheroprone flow and hyperlipidemia induce and activate TIFA in vitro and in vivo. For the priming of signal 1, sterol regulatory element-binding protein 2 transactivates TIFA, which in turn induces NF-κB activation and augments the transcription of NLRP3 inflammasome components. For the activation of signal 2, Akt is involved in TIFA Thr9 phosphorylation, which is essential for TIFA-TIFA homophilic oligomerization. Thr9 phosphorylation-dependent TIFA oligomerization facilitates the higher-order assembly of NLRP3 inflammasome, as indicated by the interaction between TIFA and caspase-1 in the activated ECs. Our results suggest that TIFA is a crucial mediator in the endothelial innate immune response by potentiating and amplifying NLRP3 inflammasome via augmenting signals 1 and 2.

Krüppel-Like Factor 4 Regulation of Cholesterol-25-Hydroxylase and Liver X Receptor Mitigates Atherosclerosis Susceptibility.

BACKGROUND: Atherosclerosis is a multifaceted inflammatory disease involving cells in the vascular wall (eg, endothelial cells [ECs]), as well as circulating and resident immunogenic cells (eg, monocytes/macrophages). Acting as a ligand for liver X receptor (LXR), but an inhibitor of SREBP2 (sterol regulatory element-binding protein 2), 25-hydroxycholesterol, and its catalyzing enzyme cholesterol-25-hydroxylase (Ch25h) are important in regulating cellular inflammatory status and cholesterol biosynthesis in both ECs and monocytes/macrophages. METHODS: Bioinformatic analyses were used to investigate RNA-sequencing data to identify cholesterol oxidation and efflux genes regulated by Krüppel-like factor 4 (KLF4). In vitro experiments involving cultured ECs and macrophages and in vivo methods involving mice with Ch25h ablation were then used to explore the atheroprotective role of KLF4-Ch25h/LXR. RESULTS: Vasoprotective stimuli increased the expression of Ch25h and LXR via KLF4. The KLF4-Ch25h/LXR homeostatic axis functions through suppressing inflammation, evidenced by the reduction of inflammasome activity in ECs and the promotion of M1 to M2 phenotypic transition in macrophages. The increased atherosclerosis in apolipoprotein E-/-/Ch25h-/- mice further demonstrates the beneficial role of the KLF4-Ch25h/LXR axis in vascular function and disease. CONCLUSIONS: KLF4 transactivates Ch25h and LXR, thereby promoting the synergistic effects between ECs and macrophages to protect against atherosclerosis susceptibility.

MicroRNA-92a Mediates Endothelial Dysfunction in CKD.

CKD is an independent risk factor for cardiovascular disease (CVD). The accumulation of uremic toxins in CKD induces oxidative stress and endothelial dysfunction. MicroRNA-92a (miR-92a) is induced by oxidative stress in endothelial cells (ECs) and involved in angiogenesis and atherosclerosis. We investigated a role for oxidative stress-responsive miR-92a in CKD. Our study of patients at three clinical sites showed increased serum miR-92a level with decreased kidney function. In cultured ECs, human CKD serum or uremic toxins (such as indoxyl sulfate), compared with non-CKD serum, induced the levels of miR-92a and suppressed the expression of miR-92a targets, including key endothelial-protective molecules. The antioxidant N-acetylcysteine inhibited these vasculopathic properties. In rats, adenine-induced CKD associated with increased levels of miR-92a in aortas, serum, and CD144+ endothelial microparticles. Furthermore, CD144+ microparticles from human uremic serum contained more miR-92a than those from control serum. Additional analysis showed a positive correlation between serum levels of miR-92a and indoxyl sulfate in a cohort of patients with ESRD undergoing hemodialysis. Collectively, our findings suggest that the uremic toxins accumulated in CKD can upregulate miR-92a in ECs, which impairs EC function and predisposes patients to CVD.

Oxidative stress activates endothelial innate immunity via sterol regulatory element binding protein 2 (SREBP2) transactivation of microRNA-92a.

BACKGROUND: Oxidative stress activates endothelial innate immunity and disrupts endothelial functions, including endothelial nitric oxide synthase-derived nitric oxide bioavailability. Here, we postulated that oxidative stress induces sterol regulatory element-binding protein 2 (SREBP2) and microRNA-92a (miR-92a), which in turn activate endothelial innate immune response, leading to dysfunctional endothelium. METHODS AND RESULTS: Using cultured endothelial cells challenged by diverse oxidative stresses, hypercholesterolemic zebrafish, and angiotensin II-infused or aged mice, we demonstrated that SREBP2 transactivation of microRNA-92a (miR-92a) is oxidative stress inducible. The SREBP2-induced miR-92a targets key molecules in endothelial homeostasis, including sirtuin 1, Krüppel-like factor 2, and Krüppel-like factor 4, leading to NOD-like receptor family pyrin domain-containing 3 inflammasome activation and endothelial nitric oxide synthase inhibition. In endothelial cell-specific SREBP2 transgenic mice, locked nucleic acid-modified antisense miR-92a attenuates inflammasome, improves vasodilation, and ameliorates angiotensin II-induced and aging-related atherogenesis. In patients with coronary artery disease, the level of circulating miR-92a is inversely correlated with endothelial cell-dependent, flow-mediated vasodilation and is positively correlated with serum level of interleukin-1ß. CONCLUSIONS: Our findings suggest that SREBP2-miR-92a-inflammasome exacerbates endothelial dysfunction during oxidative stress. Identification of this mechanism may help in the diagnosis or treatment of disorders associated with oxidative stress, innate immune activation, and endothelial dysfunction.

AMPKα2 exerts its anti-inflammatory effects through PARP-1 and Bcl-6.

B-cell lymphoma-6 protein (Bcl-6) is a corepressor for inflammatory mediators such as vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 and -3, which function to recruit monocytes to vascular endothelial cells upon inflammation. Poly [ADP ribose] polymerase 1 (PARP-1) is proinflammatory, in part through its binding at the Bcl-6 intron 1 to suppress Bcl-6 expression. We investigated the mechanisms by which PARP-1 dissociates from the Bcl-6 intron 1, ultimately leading to attenuation of endothelial inflammation. Analysis of the PARP-1 primary sequence suggested that phosphorylation of PARP-1 Serine 177 (Ser-177) by AMP-activated protein kinase (AMPK) is responsible for the induction of Bcl-6. Our results show that AMPK activation with treatment of 5-aminoimidazole-4-carboxamide ribonucleotide, metformin, or pulsatile shear stress induces PARP-1 dissociation from the Bcl-6 intron 1, increases Bcl-6 expression, and inhibits expression of inflammatory mediators. Conversely, AMPKα suppression or knockdown produces the opposite effects. The results demonstrate an anti-infamatory pathway linking AMPK, PARP-1, and Bcl-6 in endothelial cells.

Endothelial dysfunction: the role of sterol regulatory element-binding protein-induced NOD-like receptor family pyrin domain-containing protein 3 inflammasome in atherosclerosis.

PURPOSE OF REVIEW: Great effort has been devoted to elucidate the molecular mechanisms by which inflammasome in macrophages contributes to atherosclerosis. Inflammasome in vascular endothelial cells and its causal relationship with endothelial dysfunction in atherosclerosis are less understood. Here, we review the recent studies of inflammasome and its activation in endothelial cells, and highlight such endothelial inflammatory response in atherosclerosis. RECENT FINDINGS: Inflammasomes are critical effectors in innate immunity, and their activation in macrophages and the arterial wall contributes to atherogenesis. Sterol regulatory element-binding protein 2, a master regulator in cholesterol biosynthesis, can be activated in a noncanonical manner, which leads to the activation of the NOD-like receptor family pyrin domain-containing protein inflammasome in macrophages and endothelial cells. Results from in-vitro and in-vivo models suggest that sterol regulatory element-binding protein 2 is a key molecule in aggravating proinflammatory responses in endothelial cells and promoting atherosclerosis. SUMMARY: The SREBP-induced NOD-like receptor family pyrin domain-containing protein inflammasome and its instigation of innate immunity is an important contributor to atherosclerosis. Elucidating the underlying mechanisms will expand our understanding of endothelial dysfunction and its dynamic interaction with vascular inflammation. Furthermore, targeting SREBP-inflammasome pathways can be a therapeutic strategy for attenuating atherosclerosis.

Development of an iPSC-derived tissue-resident macrophage-based platform for the in vitro immunocompatibility assessment of human tissue engineered matrices.

Upon implanting tissue-engineered heart valves (TEHVs), blood-derived macrophages are believed to orchestrate the remodeling process. They initiate the immune response and mediate the remodeling of the TEHV, essential for the valve's functionality. The exact role of another macrophage type, the tissue-resident macrophages (TRMs), has not been yet elucidated even though they maintain the homeostasis of native tissues. Here, we characterized the response of hTRM-like cells in contact with a human tissue engineered matrix (hTEM). HTEMs comprised intracellular peptides with potentially immunogenic properties in their ECM proteome. Human iPSC-derived macrophages (iMφs) could represent hTRM-like cells in vitro and circumvent the scarcity of human donor material. iMφs were derived and after stimulation they demonstrated polarization towards non-/inflammatory states. Next, they responded with increased IL-6/IL-1ß secretion in separate 3/7-day cultures with longer production-time-hTEMs. We demonstrated that iMφs are a potential model for TRM-like cells for the assessment of hTEM immunocompatibility. They adopt distinct pro- and anti-inflammatory phenotypes, and both IL-6 and IL-1ß secretion depends on hTEM composition. IL-6 provided the highest sensitivity to measure iMφs pro-inflammatory response. This platform could facilitate the in vitro immunocompatibility assessment of hTEMs and thereby showcase a potential way to achieve safer clinical translation of TEHVs.

Combining Cell Technologies With Biomimetic Tissue Engineering Applications: A New Paradigm for Translational Cardiovascular Therapies.

Cardiovascular disease is a major cause of morbidity and mortality worldwide and, to date, the clinically available prostheses still present several limitations. The design of next-generation regenerative replacements either based on cellular or extracellular matrix technologies can address these shortcomings. Therefore, tissue engineered constructs could potentially become a promising alterative to the current therapeutic options for patients with cardiovascular diseases. In this review, we selectively present an overview of the current tissue engineering tools such as induced pluripotent stem cells, biomimetic materials, computational modeling, and additive manufacturing technologies, with a focus on their application to translational cardiovascular therapies. We discuss how these advanced technologies can help the development of biomimetic tissue engineered constructs and we finally summarize the latest clinical evidence for their use, and their potential therapeutic outcome.

Macrophage-extracellular matrix interactions: Perspectives for tissue engineered heart valve remodeling.

In situ heart valve tissue engineering approaches have been proposed as promising strategies to overcome the limitations of current heart valve replacements. Tissue engineered heart valves (TEHVs) generated from in vitro grown tissue engineered matrices (TEMs) aim at mimicking the microenvironmental cues from the extracellular matrix (ECM) to favor integration and remodeling of the implant. A key role of the ECM is to provide mechanical support to and attract host cells into the construct. Additionally, each ECM component plays a critical role in regulating cell adhesion, growth, migration, and differentiation potential. Importantly, the immune response to the implanted TEHV is also modulated biophysically via macrophage-ECM protein interactions. Therefore, the aim of this review is to summarize what is currently known about the interactions and signaling networks occurring between ECM proteins and macrophages, and how these interactions may impact the long-term in situ remodeling outcomes of TEMs. First, we provide an overview of in situ tissue engineering approaches and their clinical relevance, followed by a discussion on the fundamentals of the remodeling cascades. We then focus on the role of circulation-derived and resident tissue macrophages, with particular emphasis on the ramifications that ECM proteins and peptides may have in regulating the host immune response. Finally, the relevance of these findings for heart valve tissue engineering applications is discussed.

Human iPSCs and Genome Editing Technologies for Precision Cardiovascular Tissue Engineering.

Induced pluripotent stem cells (iPSCs) originate from the reprogramming of adult somatic cells using four Yamanaka transcription factors. Since their discovery, the stem cell (SC) field achieved significant milestones and opened several gateways in the area of disease modeling, drug discovery, and regenerative medicine. In parallel, the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) revolutionized the field of genome engineering, allowing the generation of genetically modified cell lines and achieving a precise genome recombination or random insertions/deletions, usefully translated for wider applications. Cardiovascular diseases represent a constantly increasing societal concern, with limited understanding of the underlying cellular and molecular mechanisms. The ability of iPSCs to differentiate into multiple cell types combined with CRISPR-Cas9 technology could enable the systematic investigation of pathophysiological mechanisms or drug screening for potential therapeutics. Furthermore, these technologies can provide a cellular platform for cardiovascular tissue engineering (TE) approaches by modulating the expression or inhibition of targeted proteins, thereby creating the possibility to engineer new cell lines and/or fine-tune biomimetic scaffolds. This review will focus on the application of iPSCs, CRISPR-Cas9, and a combination thereof to the field of cardiovascular TE. In particular, the clinical translatability of such technologies will be discussed ranging from disease modeling to drug screening and TE applications.

Role of endothelial cells in pulmonary fibrosis via SREBP2 activation.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited treatment options. Despite endothelial cells (ECs) comprising 30% of the lung cellular composition, the role of EC dysfunction in pulmonary fibrosis (PF) remains unclear. We hypothesize that sterol regulatory element-binding protein 2 (SREBP2) plays a critical role in the pathogenesis of PF via EC phenotypic modifications. Transcriptome data demonstrate that SREBP2 overexpression in ECs led to the induction of the TGF, Wnt, and cytoskeleton remodeling gene ontology pathways and the increased expression of mesenchymal genes, such as snail family transcriptional repressor 1 (snai1), α-smooth muscle actin, vimentin, and neural cadherin. Furthermore, SREBP2 directly bound to the promoter regions and transactivated these mesenchymal genes. This transcriptomic change was associated with an epigenetic and phenotypic switch in ECs, leading to increased proliferation, stress fiber formation, and ECM deposition. Mice with endothelial-specific transgenic overexpression of SREBP2 (EC-SREBP2[N]-Tg mice) that were administered bleomycin to induce PF demonstrated exacerbated vascular remodeling and increased mesenchymal transition in the lung. SREBP2 was also found to be markedly increased in lung specimens from patients with IPF. These results suggest that SREBP2, induced by lung injury, can exacerbate PF in rodent models and in human patients with IPF.

Endothelial mechanobiology.

Lining the luminal surface of the vasculature, endothelial cells (ECs) are in direct contact with and differentially respond to hemodynamic forces depending on their anatomic location. Pulsatile shear stress (PS) is defined by laminar flow and is predominantly located in straight vascular regions, while disturbed or oscillatory shear stress (OS) is localized to branch points and bifurcations. Such flow patterns have become a central focus of vascular diseases, such as atherosclerosis, because the focal distribution of endothelial dysfunction corresponds to regions exposed to OS, whereas endothelial homeostasis is maintained in regions defined by PS. Deciphering the mechanotransduction events that occur in ECs in response to differential flow patterns has required the innovation of multidisciplinary approaches in both in vitro and in vivo systems. The results from these studies have identified a multitude of shear stress-regulated molecular networks in the endothelium that are implicated in health and disease. This review outlines the significance of scientific findings generated in collaboration with Dr. Shu Chien.

Intrinsic Endocardial Defects Contribute to Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a complex congenital heart disease characterized by abnormalities in the left ventricle, associated valves, and ascending aorta. Studies have shown intrinsic myocardial defects but do not sufficiently explain developmental defects in the endocardial-derived cardiac valve, septum, and vasculature. Here, we identify a developmentally impaired endocardial population in HLHS through single-cell RNA profiling of hiPSC-derived endocardium and human fetal heart tissue with an underdeveloped left ventricle. Intrinsic endocardial defects contribute to abnormal endothelial-to-mesenchymal transition, NOTCH signaling, and extracellular matrix organization, key factors in valve formation. Endocardial abnormalities cause reduced cardiomyocyte proliferation and maturation by disrupting fibronectin-integrin signaling, consistent with recently described de novo HLHS mutations associated with abnormal endocardial gene and fibronectin regulation. Together, these results reveal a critical role for endocardium in HLHS etiology and provide a rationale for considering endocardial function in regenerative strategies.

Short-Term High-Salt Diet Increases Corin Level to Regulate the Salt-Water Balance in Humans and Rodents.

BACKGROUND: Dietary sodium and potassium affect the fluctuation in blood pressure (BP) and renal function. Corin, with its enzymatic activity to convert pro-atrial natriuretic peptide (pro-ANP) to biologically active ANP, regulates BP, cardiac, and renal functions. We investigated whether corin expression responds to a high-salt (HS) diet to regulate salt and water balance. METHODS: Forty-two volunteers followed 3 sequential diets for 7 days each: a low-salt (LS) diet (3.0 g/day NaCl), a HS diet (18.0 g/day NaCl), followed by an HS diet with K+ supplementation (HS + K+) (18.0 g/day NaCl and 4.5 g/day KCl). RESULTS: Corin level was higher with the HS diet than the LS and HS + K+ diets and was positively correlated with systolic BP (SBP) and 24-hour urinary Na+ and microalbumin (U-mALB) excretion. In rodents, serum and renal levels of corin were transiently increased with the HS diet and were decreased if the HS diet was continued for up to 7 days. HS loading increased SBP, 24-hour urinary Na+, U-mALB excretion, and the expression of proprotein convertase subtilisin/kexin-6 (PCSK6), a corin activator. Knockdown of PCSK6 or corin in high salt-treated M1-cortical collecting duct (M1-CCD) cells increased the expression of aquaporin 2 (AQP2) and ß-epithelial Na+ channel (ß-ENaC). CONCLUSIONS: Short-term HS may induce the PCSK6-corin-ANP-AQP2/ß-ENaC pathway in the kidney. Enhanced serum corin level in humans and rodents is positively correlated with HS-induced SBP and 24-hour urinary Na+ and U-mALB excretion, which suggests that corin is involved in the salt-water balance in response to HS intake. CLINICAL TRIALS REGISTRATION: Public Trials Registry Number NCT02915315.