Vascular Homeostasis Research Articles

EGFR activation differentially affects the inflammatory profiles of female human aortic and coronary artery endothelial cells

Endothelial cells (EC) are key players in vascular function, homeostasis and inflammation. EC show substantial heterogeneity due to inter-individual variability (e.g. sex-differences) and intra-individual differences as they originate from different organs or vessels. This variability may lead to different responsiveness to external stimuli. Here we compared the responsiveness of female human primary EC from the aorta (HAoEC) and coronary arteries (HCAEC) to Epidermal Growth Factor Receptor (EGFR) activation. EGFR is an important signal integration hub for vascular active substances with physiological and pathophysiological relevance. Our transcriptomic analysis suggested that EGFR activation differentially affects the inflammatory profiles of HAoEC and HCAEC, particularly by inducing a HCAEC-driven leukocyte attraction but a downregulation of adhesion molecule and chemoattractant expression in HAoEC. Experimental assessments of selected inflammation markers were performed to validate these predictions and the results confirmed a dual role of EGFR in these cells: its activation initiated an anti-inflammatory response in HAoEC but a pro-inflammatory one in HCAEC. Our study highlights that, although they are both arterial EC, female HAoEC and HCAEC are distinguishable with regard to the role of EGFR and its involvement in inflammation regulation, what may be relevant for vascular maintenance but also the pathogenesis of endothelial dysfunction.

NOS3, ET1, TGF-β1, and THBS1 polymorphisms modulating clinical complications in sickle cell disease patients

Clinical manifestations are highly variable in sickle cell disease (SCD) and polymorphisms (SNP) may modify vascular homeostasis. Because of this, we investigated of SNP of NOS3, ET1, TGF-β1 and THBS1 genes in vascular complications of SCD. High risk of hospitalization was found for genotypes NOS3TC/CC (rs2070744 - RR: 1.94) and THBS1AG/GG (rs1478604 - RR: 1.59) while low risk to NOS3GT/TT (rs1799983 - RR: 0.53). Hydroxyurea treatment was more frequent in the genotypes NOS3TC/CC (rs2070744 - RR: 1.83), ET1GT/TT (rs5370 - RR: 2.67) and ET1TG/GG (rs1800541 - RR: 1.43). Alleles ET1TG/GG (rs1800541) showed correlation for abdominal, arm and leg pain, bone disorders, cholelithiasis and vaso-occlusive crisis, while ET1GT/TT (rs5370) with chest pain, painful episodes, splenic sequestration and stroke. Interestingly, the NOS3GT/TT (rs1799983) alleles had a high stroke risk (RR:4.21), though, protection against bone disorders, cholelithiasis and less risk of hospitalization. We believed that our results indicate polymorphisms that may provide benefits in the treating Clinical Complications in SCD Patients, especially in vascular SCD pathophysiology, modulating vaso-occlusion crisis.

SUMOylation and coupling of eNOS mediated by PIAS1 contribute to maintenance of vascular homeostasis.

Endothelial dysfunction (ED) is commonly considered a crucial initiating step in the pathogenesis of numerous cardiovascular diseases. The coupling of endothelial nitric oxide synthase (eNOS) is important in maintaining normal endothelial functions. However, it still remains elusive whether and how eNOS SUMOylation affects the eNOS coupling. In the study, we investigate the roles and possible action mechanisms of protein inhibitor of activated STAT 1 (PIAS1) in ED. Human umbilical vein endothelial cells (HUVECs) treated with palmitate acid (PA) invitro and ApoE-/- mice fed with high-fat diet (HFD) invivo were constructed as the ED models. Our invivo data show that PIAS1 alleviates the dysfunction of vascular endothelium by increasing nitric oxide (NO) level, reducing malondialdehyde (MDA) level, and activating the phosphatidylinositol 3-kinase-protein kinase B-endothelial nitric oxide synthase (PI3K-AKT-eNOS) signaling in ApoE-/- mice. Our invitro data also show that PIAS1 can SUMOylate eNOS under endogenous conditions; moreover, it antagonizes the eNOS uncoupling induced by PA. The findings demonstrate that PIAS1 alleviates the dysfunction of vascular endothelium by promoting the SUMOylation and inhibiting the uncoupling of eNOS, suggesting that PIAS1 would become an early predictor of atherosclerosis and a new potential target of the hyperlipidemia-related cardiovascular diseases.

Evaluating the antioxidant potential of resveratrol-gold nanoparticles in preventing oxidative stress in endothelium on a chip

Vascular endothelial cells play a vital role in the health and maintenance of vascular homeostasis, but hyperglycemia disrupts their function by increasing cellular oxidative stress. Resveratrol, a plant polyphenol, possesses antioxidant properties that can mitigate oxidative stress. Addressing the challenges of its limited solubility and stability, gold nanoparticles (GNps) were utilized as carriers. A microfluidic chip (MFC) with dynamic flow conditions was designed to simulate body vessels and to investigate the antioxidant properties of resveratrol gold nanoparticles (RGNps), citrate gold nanoparticles (CGNps), and free Resveratrol on human umbilical vein endothelial cells (HUVEC). The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was employed to measure the extracellular antioxidant potential, and cell viability was determined using the Alamar Blue test. For assessing intracellular oxidative stress, the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was conducted, and results from both the cell culture plate and MFC were compared. Free Resveratrol demonstrated peak DPPH scavenging activity but had a cell viability of about 24–35%. RGNPs, both 3.0 ± 0.5 nm and 20.2 ± 4.7 nm, consistently showed high cell viability (more than about 90%) across tested concentrations. Notably, RGNPs (20 nm) exhibited antioxidative properties through DPPH scavenging activity (%) in the range of approximately 38–86% which was greater than that of CGNps at about 21–32%. In the MFC,the DCFH-DA analysis indicated that RGNPs (20 nm) reduced cellular oxidative stress by 57–82%, surpassing both CGNps and free Resveratrol. Morphologically, cells in the MFC presented superior structure compared to those in traditional cell culture plates, and the induction of hyperglycemia successfully led to the formation of multinucleated variant endothelial cells (MVECs). The MFC provides a distinct advantage in observing cell morphology and inducing endothelial cell dysfunction. RGNps have demonstrated significant potential in alleviating oxidative stress and preventing endothelial cell disorders.

Effect of Mechanical Stimuli on the Phenotypic Plasticity of Induced Pluripotent Stem-Cell-Derived Vascular Smooth Muscle Cells in a 3D Hydrogel.

Introduction: Vascular smooth muscle cells (VSMCs) play a pivotal role in vascular homeostasis, with dysregulation leading to vascular complications. Human-induced pluripotent stem-cell (hiPSC)-derived VSMCs offer prospects for personalized disease modeling and regenerative strategies. Current research lacks comparative studies on the impact of three-dimensional (3D) substrate properties under cyclic strain on phenotypic adaptation in hiPSC-derived VSMCs. Here, we aim to investigate the impact of intrinsic substrate properties, such as the hydrogel's elastic modulus and cross-linking density in a 3D static and dynamic environment, on the phenotypical adaptation of human mural cells derived from hiPSC-derived organoids (ODMCs), compared to aortic VSMCs. Methods and results: ODMCs were cultured in two-dimensional (2D) conditions with synthetic or contractile differentiation medium or in 3D Gelatin Methacryloyl (GelMa) substrates with varying degrees of functionalization and percentages to modulate Young's modulus and cross-linking density. Cells in 3D substrates were exposed to cyclic, unidirectional strain. Phenotype characterization was conducted using specific markers through immunofluorescence and gene expression analysis. Under static 2D culture, ODMCs derived from hiPSCs exhibited a VSMC phenotype, expressing key mural markers, and demonstrated a level of phenotypic plasticity similar to primary human VSMCs. In static 3D culture, a substrate with a higher Young's modulus and cross-linking density promoted a contractile phenotype in ODMCs and VSMCs. Dynamic stimulation in the 3D substrate promoted a switch toward a contractile phenotype in both cell types. Conclusion: Our study demonstrates phenotypic plasticity of human ODMCs in response to 2D biological and 3D mechanical stimuli that equals that of primary human VSMCs. These findings may contribute to the advancement of tailored approaches for vascular disease modeling and regenerative strategies.

PDE4 Phosphodiesterases in Cardiovascular Diseases: Key Pathophysiological Players and Potential Therapeutic Targets

3′,5′-cyclic adenosine monophosphate (cAMP) is a second messenger critically involved in the control of a myriad of processes with significant implications for vascular and cardiac cell function. The temporal and spatial compartmentalization of cAMP is governed by the activity of phosphodiesterases (PDEs), a superfamily of enzymes responsible for the hydrolysis of cyclic nucleotides. Through the fine-tuning of cAMP signaling, PDE4 enzymes could play an important role in cardiac hypertrophy and arrhythmogenesis, while it decisively influences vascular homeostasis through the control of vascular smooth muscle cell proliferation, migration, differentiation and contraction, as well as regulating endothelial permeability, angiogenesis, monocyte/macrophage activation and cardiomyocyte function. This review summarizes the current knowledge and recent advances in understanding the contribution of the PDE4 subfamily to cardiovascular function and underscores the intricate challenges associated with targeting PDE4 enzymes as a therapeutic strategy for the management of cardiovascular diseases.

The Function and Mechanism Research of NLRP6 in Bone Marrow Microenvironment in Myeloproliferative Neoplasms

Since the important role of inflammation in myeloproliferative neoplasms (MPN) progression, inflammasome is an important regulator of inflammation involved in the development of tumor. Here, we aimed to investigate the function of NLRP6 in bone marrow (BM) microenvironment in MPN in the hope of getting better treatment for MPN. We first analyzed that NLRP6 was highly expressed in BM mononuclear cells from individuals newly diagnosed with ET, PV, PMF patients (with or without JAK2 V617Fmutation). Similar results were obtained in BM cells and BM stromal cells of MPL W515L transduced C57BL/6 mice (C57BL/6 MPN). The results were validated that NLRP6 of the BM microenvironment was involved in the development of MPN. We thus hypothesized that the absence of NLRP6 in BM microenvironment affects the development of MPN. NLRP6 -/- mice and C57BL/6 mice MPN models were established by the same method. The experiments demonstrated that overall survival of NLRP6 -/- MPN mice was significantly prolonged compared to C57BL/6 MPN mice. Loss of NLRP6 in BM microenvironment interfered with hematopoiesis of BM, but it had no effect on the extramedullary hematopoiesis (EMH) in spleen. Pathological analysis showed that the destruction of spleen structure in NLRP6 -/- MPN mice and the infiltration of atypical megakaryocytes were less than C57BL/6 MPN mice. The tumor load in spleen was significantly reduced and the structure of spleen was intact, the hematopoietic function of the BM returned to normal and the tumor load in the BM was significantly reduced at 5 weeks in NLRP6 -/- MPN mice after sequential transplantation. It suggested that knockout of NLRP6 in the BM microenvironment significantly attenuated the tumorigenicity of malignant HSCs, delayed or even inhibited the development of MPN disease. The expression of inflammatory cytokines, abnormal megakaryocytes and the phenomenon of aggregation and reticular fibers in BM microenvironment were significantly reduced in NLRP6 -/- MPN group compared with the control group. After sequential transplantation, the destruction of BM structure was alleviated, megakaryocytes aggregation and heteromorphic megakaryocytes in BM were reduced, and reticular fibers were significantly diminished. Furthermore, the cytokines promoting disease progression in BM microenvironment were significantly reduced in NLRP6 -/- MPN group, suggesting that knockout of NLRP6 can improve the BM microenvironment and alleviate the progression of myelofibrosis. Finally, to gain better insight into the role of NLRP6 inflammasome in MPN pathogenesis, we sought to dissect the changes with scRNA-seq and pathway identification based on available data and bioinformatic pathway analysis in NLRP6 -/- mice. The results of scRNA-seq analysis showed that C18 population of malignant HSCs of recipients in primary transplantation was added, C16, C13 and C9 cell populations disappeared after deletion of NLRP6 in BM microenvironment, while C5, C6, C7, C10 and C12 cell populations increased significantly. Regulon of various subpopulations of malignant HSCs changed significantly, mainly involving cell development and differentiation, immune response, inflammatory BM microenvironment, vascular endothelial homeostasis and tumor related genes. Our results indicate that knockout of NLRP6 in the BM microenvironment may affect the progression of MPN by altering some malignant HSCs subpopulations and regulons, participating in immune, inflammatory, tumor metabolism and other signaling pathways to regulate the functional characteristics of tumor stem cells of MPN.Our data here provide new evidence for NLRP6 as potential target in the treatment of MPN.

Liver X receptor alpha ensures blood-brain barrier function by suppressing SNAI2

In Alzheimer’s disease (AD) more than 50% of the patients are affected by capillary cerebral amyloid-angiopathy (capCAA), which is characterized by localized hypoxia, neuro-inflammation and loss of blood-brain barrier (BBB) function. Moreover, AD patients with or without capCAA display increased vessel number, indicating a reactivation of the angiogenic program. The molecular mechanism(s) responsible for BBB dysfunction and angiogenesis in capCAA is still unclear, preventing a full understanding of disease pathophysiology. The Liver X receptor (LXR) family, consisting of LXRα and LXRβ, was reported to inhibit angiogenesis and particularly LXRα was shown to secure BBB stability, suggesting a major role in vascular function. In this study, we unravel the regulatory mechanism exerted by LXRα to preserve BBB integrity in human brain endothelial cells (BECs) and investigate its role during pathological conditions. We report that LXRα ensures BECs identity via constitutive inhibition of the transcription factor SNAI2. Accordingly, deletion of brain endothelial LXRα is associated with impaired DLL4-NOTCH signalling, a critical signalling pathway involved in vessel sprouting. A similar response was observed when BECs were exposed to hypoxia, with concomitant LXRα decrease and SNAI2 increase. In support of our cell-based observations, we report a general increase in vascular SNAI2 in the occipital cortex of AD patients with and without capCAA. Importantly, SNAI2 strongly associated with vascular amyloid-beta deposition and angiopoietin-like 4, a marker for hypoxia. In hypoxic capCAA vessels, the expression of LXRα may decrease leading to an increased expression of SNAI2, and consequently BECs de-differentiation and sprouting. Our findings indicate that LXRα is essential for BECs identity, thereby securing BBB stability and preventing aberrant angiogenesis. These results uncover a novel molecular pathway essential for BBB identity and vascular homeostasis providing new insights on the vascular pathology affecting AD patients.

Sulfur signaling pathway in cardiovascular disease

Hydrogen sulfide (H2S) and sulfur dioxide (SO2), recognized as endogenous sulfur-containing gas signaling molecules, were the third and fourth molecules to be identified subsequent to nitric oxide and carbon monoxide (CO), and exerted diverse biological effects on the cardiovascular system. However, the exact mechanisms underlying the actions of H2S and SO2 have remained elusive until now. Recently, novel post-translational modifications known as S-sulfhydration and S-sulfenylation, induced by H2S and SO2 respectively, have been proposed. These modifications involve the chemical alteration of specific cysteine residues in target proteins through S-sulfhydration and S-sulfenylation, respectively. H2S induced S-sulfhydrylation can have a significant impact on various cellular processes such as cell survival, apoptosis, cell proliferation, metabolism, mitochondrial function, endoplasmic reticulum stress, vasodilation, anti-inflammatory response and oxidative stress in the cardiovascular system. Alternatively, S-sulfenylation caused by SO2 serves primarily to maintain vascular homeostasis. Additional research is warranted to explore the physiological function of proteins with specific cysteine sites, despite the considerable advancements in comprehending the role of H2S-induced S-sulfhydration and SO2-induced S-sulfenylation in the cardiovascular system. The primary objective of this review is to present a comprehensive examination of the function and potential mechanism of S-sulfhydration and S-sulfenylation in the cardiovascular system. Proteins that undergo S-sulfhydration and S-sulfenylation may serve as promising targets for therapeutic intervention and drug development in the cardiovascular system. This could potentially expedite the future development and utilization of drugs related to H2S and SO2.

Effects of Mechanical Stress on Endothelial Cells In Situ and In Vitro.

Endothelial cells lining blood vessels are essential for maintaining vascular homeostasis and mediate several pathological and physiological processes. Mechanical stresses generated by blood flow and other biomechanical factors significantly affect endothelial cell activity. Here, we review how mechanical stresses, both in situ and in vitro, affect endothelial cells. We review the basic principles underlying the cellular response to mechanical stresses. We also consider the implications of these findings for understanding the mechanisms of mechanotransducer and mechano-signal transduction systems by cytoskeletal components.

RGS5 maintaining vascular homeostasis is altered by the tumor microenvironment

BackgroundRegulator of G protein signaling 5 (RGS5), as a negative regulator of G protein-coupled receptor (GPCR) signaling, is highly expressed in arterial VSMCs and pericytes, which is involved in VSMC phenotypic heterogeneity and vascular remodeling in tumors. However, its role in normal and tumor vascular remodeling is controversial.MethodsRGS5 knockout (Rgs5-KO) mice and RGS5 overexpression or knockdown in VSMCs in vivo by adeno-associated virus type 9 (AAV) carrying RGS5 cDNA or small hairpin RNA (shRNA) targeting RGS5 were used to determine the functional significance of RGS5 in vascular inflammation. RGS5 expression in the triple-negative (TNBCs) and non-triple-negative breast cancers (Non-TNBCs) was determined by immunofluorescent and immunohistochemical staining. The effect of breast cancer cell-conditioned media (BC-CM) on the pro-inflammatory phenotype of VSMCs was measured by phagocytic activity assays, adhesion assay and Western blot.ResultsWe identified that knockout and VSMC-specific knockdown of RGS5 exacerbated accumulation and pyroptosis of pro-inflammatory VSMCs, resulting in vascular remodeling, which was negated by VSMC-specific RGS5 overexpression. In contrast, in the context of breast cancer tissues, the role of RGS5 was completely disrupted. RGS5 expression was increased in the triple-negative breast cancer (TNBC) tissues and in the tumor blood vessels, accompanied with an extensive vascular network. VSMCs treated with BC-CM displayed enhanced pro-inflammatory phenotype and higher adherent with macrophages. Furthermore, tumor-derived RGS5 could be transferred into VSMCs.ConclusionsThese findings suggest that tumor microenvironment shifts the function of RGS5 from anti-inflammation to pro-inflammation and induces the pro-inflammatory phenotype of VSMCs that is favorable for tumor metastasis.

Mechanisms of Endothelial Cell Membrane Repair: Progress and Perspectives.

Endothelial cells are the crucial inner lining of blood vessels, which are pivotal in vascular homeostasis and integrity. However, these cells are perpetually subjected to a myriad of mechanical, chemical, and biological stresses that can compromise their plasma membranes. A sophisticated repair system involving key molecules, such as calcium, annexins, dysferlin, and MG53, is essential for maintaining endothelial viability. These components orchestrate complex mechanisms, including exocytosis and endocytosis, to repair membrane disruptions. Dysfunctions in this repair machinery, often exacerbated by aging, are linked to endothelial cell death, subsequently contributing to the onset of atherosclerosis and the progression of cardiovascular diseases (CVD) and stroke, major causes of mortality in the United States. Thus, identifying the core machinery for endothelial cell membrane repair is critically important for understanding the pathogenesis of CVD and stroke and developing novel therapeutic strategies for combating CVD and stroke. This review summarizes the recent advances in understanding the mechanisms of endothelial cell membrane repair. The future directions of this research area are also highlighted.

Specialized Retinal Endothelial Cells Modulate Blood-Retina Barrier in Diabetic Retinopathy.

Endothelial cells (EC) play essential roles in retinal vascular homeostasis. This study aimed to characterize retinal EC heterogeneity and functional diversity using single-cell RNA sequencing. Systematic analysis of cellular compositions and cell-cell interaction networks identified a unique EC cluster with high inflammatory gene expression in diabetic retina; sphingolipid metabolism is a prominent aspect correlated with changes in retinal function. Among sphingolipid-related genes, alkaline ceramidase 2 (ACER2) showed the most significant increase. Plasma samples of patients with non-proliferative diabetic retinopathy (NPDR) with or without diabetic macular edema (DME) and active PDR were collected for mass spectrometry analysis. Metabolomic profiling revealed that the ceramide levels were significantly elevated in NPDR-NDME/DME and further increased in active PDR compared with control patients. In vitro analyses showed that ACER2 overexpression retarded endothelial barrier breakdown induced by ceramide, while silencing of ACER2 further disrupted the injury. Moreover, intravitreal injection of the recombinant ACER2 adeno-associated virus rescued diabetes-induced vessel leakiness, inflammatory response, and neurovascular disease in diabetic mouse models. Together, this study revealed a new diabetes-specific retinal EC population and a negative feedback regulation pathway that reduces ceramide content and endothelial dysfunction by upregulating ACER2 expression. These findings provide insights into cell-type targeted interventions for diabetic retinopathy.

The Emerging Therapeutic Role of Prostaglandin E2 Signaling in Pulmonary Hypertension.

Mild-to-moderate pulmonary hypertension (PH) is a common complication of chronic obstructive pulmonary disease (COPD). It is characterized by narrowing and thickening of the pulmonary arteries, resulting in increased pulmonary vascular resistance (PVR) and ultimately leading to right ventricular dysfunction. Pulmonary vascular remodeling in COPD is the main reason for the increase of pulmonary artery pressure (PAP). The pathogenesis of PH in COPD is complex and multifactorial, involving chronic inflammation, hypoxia, and oxidative stress. To date, prostacyclin and its analogues are widely used to prevent PH progression in clinical. These drugs have potent anti-proliferative, anti-inflammatory, and stimulating endothelial regeneration properties, bringing therapeutic benefits to the slowing, stabilization, and even some reversal of vascular remodeling. As another well-known and extensively researched prostaglandins, prostaglandin E2 (PGE2) and its downstream signaling have been found to play an important role in various biological processes. Emerging evidence has revealed that PGE2 and its receptors (i.e., EP1-4) are involved in the regulation of pulmonary vascular homeostasis and remodeling. This review focuses on the research progress of the PGE2 signaling pathway in PH and discusses the possibility of treating PH based on the PGE2 signaling pathway.

EPCO-48. THE SINGLE-CELL AND SPATIAL TRANSCRIPTOMIC ARCHITECTURE OF MENINGEAL SOLITARY FIBROUS TUMORS PHENOCOPIES CEREBRAL VASCULAR DEVELOPMENT AND HOMEOSTASIS

Abstract INTRODUCTION Meningeal solitary fibrous tumors (SFTs) are rare mesenchymal neoplasms that are associated with local recurrence and distant metastasis. The cell states and spatial transcriptomic architecture of SFTs are unknown. METHODS Single-cell RNA sequencing or spatial transcriptomic sequencing were performed on 40,022 cells or 23,682 spatial transcriptomes from 12 meningeal SFT samples. Datasets were corrected for batch effects using Harmony and integrated using uniform maniform approximation and projection (UMAP). Clusters were defined using automated cell type classification, cell signature gene sets, cell cycle analysis, and differentially expressed marker genes. Trajectory analyses were performed using RNA velocity and pseudotime. Cell-cell communication analysis was performed using CellChat. RESULTS were validated using immunofluorescence and immunohistochemistry and generalized using comparisons to 30,934 single-cell transcriptomes from 6 meningioma samples, 139,134 perinatal human brain vascular single-cell transcriptomes, 84,138 adult human brain vascular single-cell transcriptomes, and DNA methylation profiles from 8 SFT and 221 meningioma samples. RESULTS Unsupervised hierarchical clustering of DNA methylation profiles revealed molecular distinction of meningeal SFTs from meningiomas. Deconvolution of vascular single-cell types showed SFT cell clusters resembling perinatal or adult fibroblasts, perinatal mitotic endothelia, and adult venous or arterial endothelia. UMAP and trajectory analyses showed SFTs were comprised of 8 interchangeable tumor cell states that were associated with cell adhesion (VCAM1, NCAM2), cell stress (EGR1), cell signaling (NOTCH3), extracellular matrix remodeling (ECM) (ADAMTS6, PLCG2), or protein synthesis pathways (RPL27A, RPL37). Cell-cell communication analyses identified single-cell and spatial interactions between VCAM1- or NCAM2-expressing SFT cells and endothelia or immature neurons in the tumor microenvironment, respectively. Single-cell deconvolution demonstrated evolution of ECM and protein synthesis SFT cells in paired primary/recurrent samples, and evolution of ECM SFT cells and macrophages in paired primary/metastatic samples. CONCLUSIONS The single-cell and spatial transcriptomic architecture of meningeal SFT reveals dynamic cell states that phenocopy cerebral vascular development and homeostasis.

The Role of Fibrinogen, Homocysteine and Metabolic Syndrome's Alterations in Sudden Sensorineural Hearing Loss (SSHL): A Narrative Review.

Fibrinogen and homocysteine (HCY) are molecules known to play a role in vascular homeostasis, and their blood levels are often elevated in patients with metabolic syndrome. Recent evidence suggests that sudden sensorineural hearing loss (SSHL) may have a vascular origin. This has led many authors to advocate that fibrinogen, homocysteine, and metabolic syndrome (MetS) may play a direct role in SSHL. The aim of this brief review is to examine the role and influence of these molecules and MetS on the mechanisms of SSHL. Elevated fibrinogen levels have been associated with a worse prognosis in SSHL, possibly due to increased blood viscosity and decreased blood flow. Similarly, HCY has been associated with vascular damage, particularly in hyperhomocysteinemia, although the exact association with SSHL remains controversial. MetS has been demonstrated to function both as a causative factor and as a contributor to poorer recovery in cases of SSHL. However, although some studies suggest a possible role for these biomarkers and MetS in the prognosis and treatment of SSHL, specific therapeutic and preventive strategies based solely on these factors have yet to be developed. Given their potential role in prognosis and treatment and the global epidemic of metabolic syndrome, this issue needs to be analyzed comprehensively. Thus, further quality studies need to be conducted, even though it is difficult to determine the actual impact of MetS on the development of SSHL, as it is a multifactorial disease affecting multiple organs.

Protective role of CXCR7 activation in neonatal hyperoxia-induced systemic vascular remodeling and cardiovascular dysfunction in juvenile rats

Neonatal hyperoxia induces long-term systemic vascular stiffness and cardiovascular remodeling, but the mechanisms are unclear. Chemokine receptor 7 (CXCR7) represents a key regulator of vascular homeostasis and repair by modulating TGF-β1 signaling. This study investigated whether pharmacological CXCR7 agonism prevents neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction in juvenile rats. Newborn Sprague Dawley rat pups assigned to room air or hyperoxia (85% oxygen), received CXCR7 agonist, TC14012 or placebo for 3 weeks. These rat pups were maintained in room air until 6 weeks when aortic pulse wave velocity doppler, cardiac echocardiography, aortic and left ventricular (LV) fibrosis were assessed. Neonatal hyperoxia induced systemic vascular stiffness and cardiac dysfunction in 6-week-old rats. This was associated with decreased aortic and LV CXCR7 expression. Early treatment with TC14012, partially protected against neonatal hyperoxia-induced systemic vascular stiffness and improved LV dysfunction and fibrosis in juvenile rats by decreasing TGF-β1 expression. In vitro, hyperoxia-exposed human umbilical arterial endothelial cells and coronary artery endothelial cells had increased TGF-β1 levels. However, treatment with TC14012 significantly reduced the TGF-β1 levels. These results suggest that dysregulation of endothelial CXCR7 signaling may contribute to neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction.

Abstract 13842: A Macromolecule-Linked Prodrug of Rapamycin Shows Potent Growth Inhibitory Effects Against Activated Fibroblasts Mimicking Fibroproliferative Transformation in Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) is a life-threatening disease characterized by a progressive loss of vascular cell homeostasis with an irreversible shift toward the activated proliferative phenotype. New drug delivery approaches with improved efficacy are urgently needed for better clinical management of the disease. The present studies evaluated a novel prodrug-based strategy designed to durably suppress PH through enhanced delivery of rapamycin. We hypothesized that a long-circulating rapamycin prodrug constructed with a bioeliminable polyethylene glycol (PEG) scaffold would show efficacy as a new anti-PH drug candidate in cell culture and animal model studies. Methods: A new carrier-linked prodrug was synthesized by reversibly attaching rapamycin to 4-arm PEG (PEG-[RAPA] 4 , Mn = 40 kDa). Cell growth inhibitory effects of the construct were studied in vitro with proliferative fetal lung fibroblasts (HEL299) and transformed fibroblast-type cells (SK-LMS1), and in vivo using SK-LMS1 xenografts established in athymic nude mice. The results were analyzed by comparing the growth curve slopes (one-way ANOVA with the Tukey post hoc test). Results: Rapamycin (1-5 μM) potently inhibited proliferation of both fetal and transformed fibroblasts (75-95% inhibition 7 days post-treatment) in PDGF-BB-stimulated growth conditions (40 ng/ml, 2% FBS). Serum-stimulated (10% FBS) growth of both cell lines was also suppressed with remarkable potency at all tested concentrations (≥50% and ≥65% inhibition for HEL299 and SK-LMS1, respectively). In animal model studies, PEG-[RAPA] 4 given 2х/week strongly mitigated disease progression throughout the 4-week treatment period, whereas all animals in ‘no treatment’ and ‘blank PEG’ control groups were eliminated after reaching the end point. Statistical analysis confirmed markedly delayed growth of SK-LMS1 xenografts in the prodrug group (p = 0.032). Conclusions: Polymeric carrier-linked prodrug delivery of rapamycin effectively inhibits growth of activated fibroblasts in vitro and in a preclinical model recapitulating the altered vascular cell phenotype and proliferative character of the human disease. PEG-[RAPA] 4 is a promising candidate for further evaluation as a potential therapy for PH.

Abstract 12750: Three Dimensional Environment for Efficient and Large Scale Generation of Smooth Muscle Cells From HiPSCs

Introduction: Smooth muscle cells (SMCs) play an important role in maintaining vascular homeostasis and the development of diseases. We assess feasibility and efficacy of differentiation human induced pluripotent stem cells (hiPSCs) to SMCs in a 3-dimension (3D). Methods: hiPSCs were cultured in shaker flasks or PBS-0.1 Vessels for 48 h. Differentiation initiated (as day 0) by culturing hiPSC spheroids in RPMI+B27 without insulin (B27-) and CHIR99021 (CHIR) for 24 h. Day 1, medium was replaced with RPMI+B27-, platelet derived growth factor β (PDGF-β), and transforming growth factor β1 (TGFβ1) for 48 h, refreshed medium on day 3 for 48 h. Day 5, differentiating hiPSC-spheroids were dissociated to single cells and cultured as monolayer in RPMI +4% FBS, PDGF-β, and TGFβ1 for 72 h. Refreshed medium day 8 for 72 h. Differentiation efficiency was determined by flow cytometry and fluorescence immunostaining. The biological function of 3D differentiated SMCs was assessed in vitro and in a mouse model of ischemic limb disease in vivo . Results: Both 3D differentiation methods efficiently differentiated hiPSCs into SMCs. Flow cytometry showed that 99.4% cells expressed smooth muscle actin (SMA), 97.9% expressed myosin heavy chain 11 (MYH11), 95.1% expressed SM22, 97.5% expressed calponin, and 97.7% expressed calveolin-3 using shaker flasks. They were 99.6% for SMA, 97.9% for MYH11, 97% for SM22, 89.6% for calponin, and 87.2% for calveolin-3, respectively, using PBS-0.1 Vessels. Mean SMC cell number obtained was 4.56 x 10 7 for shaker flasks and 5.69 x 10 7 for PBS-0.1 Vessels, respectively, which were significantly higher than hiPSCs differentiated in monolayer (5.7x10 6 ). Fluorescence immunostaining confirmed that SMCs differentiated from both methods expressed SMA, MYH11, SM22, Calponin, and calveolin-3. Differentiated SMCs migrated in scratch assay, contracted in response to U-46619, an analog of the endoperoxide prostaglandin, and formed tubular structure on Matrigel in vitro. Fluorescence angiogram imaging showed that SMC transplantation effectively induced perfusion in mouse ischemic limbs. Conclusions: Both 3D differentiation methods, using shaker flasks or PBS-0.1 vessels, efficiently differentiated hiPSCs into large scales of functional SMCs.

Abstract 11891: Pulmonary Delivery of Novel BEPI/NO-Microparticle for Treatment of Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is an incurable disease characterized by elevated blood pressure in the pulmonary artery causing right heart failure and even sudden cardiac death. Nitric oxide (NO) is a strong potent regulator of PAH. However, the therapeutic effects of exogenous NO depend on its concentration and its half-life is very short. Hypothesis: we prepared NO-releasing inhalers with controlled porous structures to modulate the deposition in lung. Methods and Results: NO-releasing inhalers with micro-sized pores showed improved nebulization efficiency. Furthermore, the cGMP expression and macrophage polarization were evaluated. The fluorescence intensity of Nile red was analyzed using photoluminescence spectroscopy to confirm the amounts of NO-MPs on the slide glass. As with confocal images, 0.8k BPEI/NO-MPs could arrive the lower region than 25k BPEI/NO-MPs. NO produced by endothelial nitric oxide synthase (eNOS) induce vasodilation by increasing cGMP in vascular smooth muscle cells. Hence, the delivery of NO mimicking the physical production of the endothelium in range of 0.05-0.4 nmol cm -2 min -1 is required to restore vascular homeostasis. Therefore, BPEI0.8k/NO-MPs release 0.277 nmol cm -2 min -1 of NO that mimics the environment in healthy endothelium, while BPEI25k/NO-MPs showed a higher concentration as 0.548 nmol cm -2 min -1 . BPEI0.8k/NO-MPs increased the synthesis of cGMP in cells, whereas BPEI25k/NO-MPs presented adverse effects due to an initial burst release. When cell populations were quantified, BPEI0.8k/NO-MPs increased the number of M2-like cells while decreasing the number of M1-like cells. Furthermore, using fluorescence activated cell sorting analysis, proteins and gene levels of cells, anti-inflammatory factors increased in BPEI0.8k/NO-MPs treatment groups Conclusions: novel BPEI/NO-releasing inhalers could deliver NO to deep lungs and has the potential for treatment of PAH