Mesenteric Endothelial Cells Research Articles

Abstract P125: In an Autocrine Mechanism, Endothelial Progranulin Regulates Vascular Tone and Blood Pressure via AMPK Activation

Dysfunctional endothelial cells (ECs) contribute to the development of hypertension (HTN). Progranulin (PGRN) is a secreted protein that plays a major role in the inflammatory response. We recently demonstrated that HTN is associated with increased circulating PGRN, whereas the global lack of PGRN elicits vascular dysfunction and raises blood pressure (BP). However, the cellular origin of PGRN and the molecular mechanisms by which PGRN regulates vascular tone and BP are still to be elucidated. We hypothesized that ECs secrete PGRN, which regulates the vascular phenotype and BP in an autocrine manner. We used mice with tamoxifen-inducible, Cdh5-CreERT2-mediated deletion of PGRN in ECs (PGRN EC-/-) and their counterparts as controls (PGRN EC+/+). The effectiveness of endothelial PGRN depletion was validated using en face immunostaining in the aorta and qPCR in ECs from mesenteric arteries (MA). PGRN EC-/- mice exhibited a 2.5-fold reduction in circulating PGRN levels followed by EC dysfunction in MA characterized by reduced maximal response (Emax) and sensitivity (pD2) to acetylcholine [Emax (%): PGRN EC+/+: 99.3 ± 0.7 vs PGRN EC-/-: 95.9 ± 0.5* and pD2: PGRN EC+/+: 8.1 ± 0.2 vs PGRN EC-/-: 7.0 ± 0.3*, *P<0.05]. Supplementation with recombinant PGRN (20 μg/day) normalized endothelial function. Moreover, under baseline conditions, BP (analyzed by telemetry) was slightly higher in PGRN EC-/- mice [MAP (mmHg): PGRN+/+: 110.6 ± 1.6 vs PGRN-/-: 114.3 ± 1.2]. However, treatment with a subpressor dose of angiotensin II (120 ng/kg/day for 2 weeks) only increased BP in PGRN EC-/- mice. Mechanistically, we found that knockdown of PGRN (via siRNA) in cultured mesenteric ECs (MECs) suppressed PGRN secretion, AMPK phosphorylation, and nitric oxide (NO) formation. Conversely, MECs overexpressing PGRN (via lentivirus infection) led to exacerbated PGRN secretion followed by AMPK activation and NO formation, which were blunted by blocking AMPK or EphrinA2 (PGRN receptor) with dorsomorphin (10 μM) or ALWII4127 (5 μM), respectively. Finally, endothelial-specific AMPK-deficient mice exhibited increased BP and endothelial dysfunction in MA; such effects were not affected by PGRN treatment, suggesting that AMPK is a downstream target for PGRN. In summary, our data indicate that ECs are important sources of PGRN, which supports the maintenance of the EC phenotype and BP via EphrinA2, AMPK, and NO formation. This pathway occurs in an autocrine-dependent manner.

Abstract 59: Beclin-1-Dependent Autophagy Prevents Hyperaldosternism-Induced Endothelial Dysfunction And Hypertension

Hyperaldosteronism (HA), characterized by the overproduction of aldosterone (Aldo), leads to cardiovascular damage, including endothelial dysfunction. Beclin-1 (Becn1) is a key protein regulating autophagy, a recycling process for unwanted components. Whether HA disturbs autophagic flux in endothelial cells via Becn1 is unknown. We hypothesize that Becn1-dependent autophagic flux will improve endothelial function and inhibit the hypertension (HTN) in HA. 10-to-12-week-old male C57BL/6J (WT) or Becn1 knock-in mice (Becn1 Tg ) were infused with aldosterone for 14 days (600 µg/kg/day). Vascular function was studied in mesenteric arteries (MA) and blood pressure was analyzed by radiotelemetry. HA disrupted MA autophagic flux characterized by accumulation of LC3A/B and p62 and reduction in Becn1 expression and activity followed by suppressed acetylcholine (ACh)-induced vasodilation (% relaxation) in WT [WT: 91.5±1.1 vs HA: 56.7±2.7], whereas Becn1 Tg mice were protected from HA-induced HTN [mean blood pressure (mmHg) – WT_Aldo: 149.1±1.2 vs BCN1_Aldo: 118.1±0.8] and endothelial dysfunction [HA_Becn1 Tg : 92.8±2.9]. Given the link between the immune system and cardiovascular diseases, we investigated whether HA reduces autophagic flux and BCN1 in immune cells. HA led to the accumulation of LC3A/B and p62, and decreased BCN1 activity in peritoneal cavity cells of WT mice, however bone marrow transplant from Becn1 Tg mice into WT mice did not confer protection against HA-induced changes in vascular reactivity [WT to WT_HA: 51.2±1.3 vs Becn1 Tg to WT_HA: 53.8±1.9]. As a pharmacological approach, we accelerated autophagic flux by treating mice with spermidine (3 mM for 7 days), which subsequently improved endothelial function in HA [HA: 52.7±3.9 vs HA+spermidine: 88.9±3.9]. To assess the direct impact of Aldo on disrupting autophagy flux in endothelial cells, we treated mesenteric endothelial cells (MEC) with Aldo (0.1 µM for 24 hours). This treatment resulted in the accumulation of LC3A/B and p62 proteins and a reduction in BCN1 activity, accompanied by an upregulation in the mRNA expression of pro-inflammatory cytokines. All these effects were prevented by spermidine (100 uM for 3h). These findings reveal a novel pathway in HA-associated endothelial dysfunction and HTN, where Aldo disrupts autophagic flux and inflammation by suppressing BCN1 activity, positioning BCN1 as a pharmacological target for cardiovascular outcomes in HA.

Rictor, an mTORC2 Protein, Regulates Murine Lymphatic Valve Formation Through the AKT-FOXO1 Signaling.

Lymphatic valves are specialized structures in collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves. Conventionally, in many cell types, AKT is phosphorylated at Ser473 by the mTORC2 (mammalian target of rapamycin complex 2). However, mTORC2 has not yet been implicated in lymphatic valve formation. In vivo and in vitro techniques were used to investigate the role of Rictor, a critical component of mTORC2, in lymphatic endothelium. Here, we showed that embryonic and postnatal lymphatic deletion of Rictor, a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels. RICTOR knockdown in human dermal lymphatic endothelial cells not only reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions but also abolished the upregulation of AKT activity and valve-forming genes in response to oscillatory shear stress. We further showed that the AKT target, FOXO1 (forkhead box protein O1), a repressor of lymphatic valve formation, had increased nuclear activity in Rictor knockout mesenteric lymphatic endothelial cells in vivo. Deletion of Foxo1 in Rictor knockout mice restored the number of valves to control levels in lymphatic vessels of the ear and mesentery. Our work identifies a novel role for RICTOR in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately enables the formation and maintenance of lymphatic valves.

NADPH oxidase 4-derived hydrogen peroxide counterbalances testosterone-induced endothelial dysfunction and migration.

High levels of testosterone (Testo) are associated with cardiovascular risk by increasing reactive oxygen species (ROS) formation. NADPH oxidases (NOX) are the major source of ROS in the vasculature of cardiovascular diseases. NOX4 is a unique isotype, which produces hydrogen peroxide (H2O2), and its participation in cardiovascular biology is controversial. So far, it is unclear whether NOX4 protects from Testo-induced endothelial injury. Thus, we hypothesized that supraphysiological levels of Testo induce endothelial NOX4 expression to attenuate endothelial injury. Human mesenteric vascular endothelial cells (HMECs) and human umbilical vein endothelial cells (HUVEC) were treated with Testo (10-7 M) with or without a NOX4 inhibitor [GLX351322 (10-4 M)] or NOX4 siRNA. In vivo, 10-wk-old C57Bl/6J male mice were treated with Testo (10 mg/kg) for 30 days to study endothelial function. Testo increased mRNA and protein levels of NOX4 in HMECs and HUVECs. Testo increased superoxide anion (O2-) and H2O2 production, which were abolished by NOX1 and NOX4 inhibition, respectively. Testo also attenuated bradykinin-induced NO production, which was further impaired by NOX4 inhibition. In vivo, Testo decreased H2O2 production in aortic segments and triggered endothelial dysfunction [decreased relaxation to acetylcholine (ACh)], which was further impaired by GLX351322 and by a superoxide dismutase and catalase mimetic (EUK134). Finally, Testo led to a dysregulated endothelial cell migration, which was exacerbated by GLX351322. These data indicate that supraphysiological levels of Testo increase the endothelial expression and activity of NOX4 to counterbalance the deleterious effects caused by Testo in endothelial function.NEW & NOTEWORTHY By inducing ROS formation, high levels of testosterone play a major role in the pathogenesis of cardiovascular disease. NOXs are the major sources of ROS in the vasculature of cardiovascular diseases. Herein, we describe a novel compensatory mechanism by showing that NOX4 is a protective oxidant enzyme and counterbalances the deleterious effects of testosterone in endothelial cells by modulating hydrogen peroxide formation.

P2Y2-P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis.

Schistosomiasis is an intravascular infectious disease that impacts over 200 million people globally. In its chronic stage, it leads to mesenteric inflammation with significant involvement of monocytes/macrophages. Endothelial cells lining the vessel lumens play a crucial role, and mount of evidence links this disease to a downregulation of endoprotective cell signaling favoring a primed and proinflammatory endothelial cell phenotype and therefore the loss of immunovascular homeostasis. One hallmark of infectious and inflammatory conditions is the release of nucleotides into the extracellular milieu, which, in turn, act as innate messengers, activating purinergic receptors and triggering cell-to-cell communication. ATP influences the progression of various diseases through P2X and P2Y purinergic receptor subtypes. Among these receptors, P2Y2 (P2Y2R) and P2X7 (P2X7R) receptors stand out, known for their roles in inflammation. However, their specific role in schistosomiasis has remained largely unexplored. Therefore, we hypothesized that endothelial P2Y2R and P2X7R could contribute to monocyte adhesion to mesenteric endothelial cells in schistosomiasis. Using a preclinical murine model of schistosomiasis associated with endothelial dysfunction and age-matched control mice, we showed that endothelial P2Y2R and P2X7R activation increased monocyte adhesion to cultured primary endothelial cells in both groups. However, a distinct upregulation of endothelial P2Y2R-driven canonical Ca2+ signaling was observed in the infected group, amplifying adhesion. In the control group, the coactivation of endothelial P2Y2R and P2X7R did not alter the maximal monocyte adhesion induced by each receptor individually. However, in the infected group, this coactivation induced a distinct upregulation of P2Y2R-P2X7R-driven canonical signaling, IL-1β release, and VCAM-1 expression, with underlying mechanisms involving inflammasome and NF-κB signaling. Therefore, current data suggest that schistosomiasis alters endothelial cell P2Y2R/P2X7R signaling during inflammation. These discoveries advance our understanding of schistosomiasis. This intricate interplay, driven by PAMP-triggered endothelial P2Y2R/P2X7R cross-talk, emerges as a potential key player in the mesenteric inflammation during schistosomiasis.

Not just fibrotic: endothelial-derived TGFβ maintains contractile function and lymphatic muscle phenotype during homeostasis.

Cell-cell communication within the lymphatic vasculature during homeostasis is incompletely detailed. Although many discoveries highlight the pathological roles of transforming growth factor-beta (TGFβ) in chronic vascular inflammation and associated fibrosis, only a small amount is known surrounding the role of TGFβ-signaling in homeostatic lymphatic function. Here, we discovered that pharmacological blockade of TGFβ receptor 1 (TGFβR1) negatively impacts rat mesenteric lymphatic vessel pumping, significantly reducing vessel contractility and surrounding lymphatic muscle coverage. We have identified mesenteric lymphatic endothelial cells themselves as a source of endogenous vascular TGFβ and that TGFβ production is significantly increased in these cells via activation of a number of functional pattern recognition receptors they express. We show that a continuous supply of TGFβ is essential to maintain the contractile phenotype of neighboring lymphatic muscle cells and support this conclusion through in vitro analysis of primary isolated lymphatic muscle cells that undergo synthetic differentiation during 2-D cell culture, a phenomenon that could be effectively rescued by supplementation with recombinant TGFβ. Finally, we demonstrate that lymphatic endothelial production of TGFβ is regulated, in part, by nitric oxide in a manner we propose is essential to counteract the pathological over-production of TGFβ. Taken together, these data highlight the essential role of homeostatic TGFβ signaling in the maintenance of lymphatic vascular function and highlight possible deleterious consequences of its inhibition.NEW & NOTEWORTHY The growth factor TGFβ is commonly associated with its pathological overproduction during tissue fibrosis rather than its homeostatic functions. We expose the lymphatic endothelium as a source of endogenous TGFβ, the impact of its production on the maintenance of surrounding lymphatic muscle cell phenotype, and internally regulated mechanisms of its production. Overall, these results highlight the intricate balance of TGFβ-signaling as an essential component of maintaining lymphatic contractile function.

Obesogenic diet disrupts tissue-specific mitochondrial gene signatures in the artery and capillary endothelium.

Endothelial cells (ECs) adapt to the unique needs of their resident tissue and metabolic perturbations, such as obesity. We sought to understand how obesity affects EC metabolic phenotypes, specifically mitochondrial gene expression. We investigated the mesenteric and adipose endothelium because these vascular beds have distinct roles in lipid homeostasis. Initially, we performed bulk RNA sequencing on ECs from mouse adipose and mesenteric vasculatures after a normal chow (NC) diet or high-fat diet (HFD) and found higher mitochondrial gene expression in adipose ECs compared with mesenteric ECs in both NC and HFD mice. Next, we performed single-cell RNA sequencing and categorized ECs as arterial, capillary, venous, or lymphatic. We found mitochondrial genes to be enriched in adipose compared with mesentery under NC conditions in artery and capillary ECs. After HFD, these genes were decreased in adipose ECs, becoming like mesenteric ECs. Transcription factor analysis revealed that peroxisome proliferator-activated receptor-γ (PPAR-γ) had high specificity in NC adipose artery and capillary ECs. These findings were recapitulated in single-nuclei RNA-sequencing data from human visceral adipose. The sum of these findings suggests that mesenteric and adipose arterial ECs metabolize lipids differently, and the transcriptional phenotype of the vascular beds converges in obesity due to downregulation of PPAR-γ in adipose artery and capillary ECs.NEW & NOTEWORTHY Using bulk and single-cell RNA sequencing on endothelial cells from adipose and mesentery, we found that an obesogenic diet induces a reduction in adipose endothelial oxidative phosphorylation gene expression, resulting in a phenotypic convergence of mesenteric and adipose endothelial cells. Furthermore, we found evidence that PPAR-γ drives this phenotypic shift. Mining of human data sets segregated based on body mass index supported these findings. These data point to novel mechanisms by which obesity induces endothelial dysfunction.

Abstract 18731: Connexin 43 Hemichannels Contribution to Ischemia-Reperfusion-Induced Hyperpermeability

Introduction: Ischemia-reperfusion (I-R) injury is a significant complication in vascular surgery or organ transplantation. I-R injury involves intracellular Ca2+ overload, nitric oxide (NO), oxidative stress. I-R injury increases endothelial vascular hyperpermeability leading to tissue edema and damage mediated by endothelial Ca2+ increase. The role of Ca2+ permeable channels in I-R is not defined. Connexin (Cx) proteins form hemichannels, allowing the exchange of molecules between intracellular and extracellular spaces. The Cx43 isoform is highly expressed in endothelial cells (EC). Endothelial Ca2+ increase promotes NO-dependent Cx43 S-nitrosylation. However, whether Cx43 hemichannels are involved in I-R-induced hyperpermeability is unknown. Hypothesis: VEGF-induced hyperpermeability causes increased NO-mediated Cx43 S-nitrosylation and opening of endothelial Cx43 hemichannels, leading to Ca2+ entry and exacerbating reperfusion damage. Methods: We used VEGF as I-R related pro-inflammatory agonist. Using primary cultures of mesenteric endothelial cells, we tested Cx43 hemichannel activity by ethidium uptake and Ca2+ influx with Fluo-4 indicator. We evaluated hyperpermeability in vitro and in vivo Results: Results indicate that VEGF stimulation increased hemichannel activity in EC, associated with a rapid rise of Ca2+ influx. Both hemichannel activation and Ca2+ increase were abolished by treatment with specific Cx43 blocker (Gap19) or blocking NO production with NG-monomethyl-L-arginine (LNMMA). Cx43 inhibition blunted VEGF-induced hyperpermeability both in vitro in primary EC culture and in vivo mouse mesenteric vascular bed. To further understand the participation of NO-mediated S-nitrosylation in the VEGF-induced hyperpermeability, we replaced cysteine 271 by serine to create a Cx43C271S mouse that fails to cause S-nitrosylation of Cx43. We demonstrated that this knockin mouse exhibits a decrease in VEGF-stimulated hyperpermeability in vivo. Conclusions: These results indicate that Cx43 hemichannels activation is critical to VEGF (I-R inflammatory environment)-induced hyperpermeability and the S-nitrosylated Cx43 is a key regulator in I-R damage. AHA support: # 932684 and #23DIVSUP1054931

Endothelial Hemoglobin Alpha Mediates Iron Regulation of Endothelial Nitric Oxide

Iron deficiency is associated with heart failure, chronic kidney disease, and chronic inflammation. All of these pathologies are associated with endothelial dysfunction and impaired nitric oxide (NO) signaling. Previous clinical studies have found iron deficiency anemia (IDA) increases endothelial NO signaling and attributed this change to the reduction in circulating hemoglobin. However, while free hemoglobin is a potent scavenger of NO, it has been shown in ex vivo preparations that endothelial NO signaling is unaffected by the presence of erythrocytes suggesting changes in circulating hemoglobin do not alter NO signaling. For these reasons we focused on endothelial iron regulation. Using single cell RNA sequencing data of isolated mesenteric and adipose endothelial cells generated by our lab, we have observed arterial endothelial cells have higher transferrin receptor expression compared to capillary, veinous, and lymphatic endothelial cells. This suggests that the arterial endothelium, which controls blood flow and is especially susceptible to endothelial dysfunction, may be particularly sensitive to changes in iron status. Additionally, our lab has previously shown the alpha chain of hemoglobin (Hbα) is expressed in the endothelium of small arteries and scavenges NO. We therefore hypothesized endothelial Hbα, rather than blood hemoglobin is responsible for the increased NO signaling in IDA. We first tested this hypothesis in a mouse model of IDA in which we were able to replete vascular iron stores. C57BL/6 mice were fed an iron deficient (2-6 ppm Fe) or nutrient matched control diet (48 ppm Fe) beginning at weaning. IDA mice underwent phlebotomies (10% blood volume) at 9 and 11 weeks old to aid in the progression of anemia. To restore vascular iron stores, a subset of IDA mice received a single i.p. injection of iron dextran (FeDex; 20 mg/kg) at 12 weeks. One week later complete blood counts were assessed, tissues were collected, and blood flow studies were performed. As expected, IDA mice had low blood hemoglobin (Con: 15.54 ± 0.43 vs. IDA: 6.04 ± 0.35 g/dL; p < 0.0001). FeDex did not rescue the anemia (8.55 ± 0.74 g/dL; p < 0.0001 vs. Con), but did rescue vascular iron stores as measured by ferritin light chain protein. To investigate NO signaling, we used laser speckle contrast imaging to measure changes in in vivo blood flow in response to the NO synthase inhibitor L-NAME. The magnitude of the response to L-NAME is an indicator of NO signaling. IDA mice exhibited an exaggerated response to L-NAME indicating increased NO signaling. Rescuing vascular iron stores with FeDex restored the L-NAME response back to control levels suggesting vascular iron stores, rather than the anemia itself increased NO signaling. In order to investigate the role of endothelial Hbα, we repeated these studies in endothelial specific Hbα knockout mice (Hba1 fl/fl-Cdh5-CreER T2+) developed by us. Lack of endothelial Hbα did not prevent the increased response to L-NAME in IDA mice, but did prevent the rescue by FeDex. These data suggest a model in which endothelial Hbα participates in regulating NO in response to iron, but not the increased NO in IDA.

Progranulin Maintains Blood Pressure and Vascular Tone Dependent on EphrinA2 and Sortilin1 Receptors and Endothelial Nitric Oxide Synthase Activation.

Background The mechanisms determining vascular tone are still not completely understood, even though it is a significant factor in blood pressure management. Many circulating proteins have a significant impact on controlling vascular tone. Progranulin displays anti-inflammatory effects and has been extensively studied in neurodegenerative illnesses. We investigated whether progranulin sustains the vascular tone that helps regulate blood pressure. Methods and Results We used male and female C57BL6/J wild type (progranulin+/+) and B6(Cg)-Grntm1.1Aidi/J (progranulin-/-) to understand the impact of progranulin on vascular contractility and blood pressure. We found that progranulin-/- mice display elevated blood pressure followed by hypercontractility to noradrenaline in mesenteric arteries, which is restored by supplementing the mice with recombinant progranulin. In ex vivo experiments, recombinant progranulin attenuated the vascular contractility to noradrenaline in male and female progranulin+/+ arteries, which was blunted by blocking EphrinA2 or Sortilin1. To understand the mechanisms whereby progranulin evokes anticontractile effects, we inhibited endothelial factors. N(gamma)-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) prevented the progranulin effects, whereas indomethacin (cyclooxygenase inhibitor) affected only the contractility in arteries incubated with vehicle, indicating that progranulin increases nitric oxide and decreases contractile prostanoids. Finally, recombinant progranulin induced endothelial nitric oxide synthase phosphorylation and nitric oxide production in isolated mesenteric endothelial cells. Conclusions Circulating progranulin regulates vascular tone and blood pressure via EphrinA2 and Sortilin1 receptors and endothelial nitric oxide synthase activation. Collectively, our data suggest that deficiency in progranulin is a cardiovascular risk factor and that progranulin might be a new therapeutic avenue to treat high blood pressure.

Recombinant VEGF-C (Cys156Ser) improves mesenteric lymphatic drainage and gut immune surveillance in experimental cirrhosis

Lymphatic vessels (LVs) are crucial for maintaining abdominal fluid homoeostasis and immunity. In cirrhosis, mesenteric LVs (mLVs) are dilated and dysfunctional. Given the established role of vascular endothelial growth factor-C (VEGF-C) in improving LVs, we hypothesised that VEGF-C treatment could ameliorate the functions of mLVs in cirrhosis. In this study, we developed a nanoformulation comprising LV-specific growth factor, recombinant human VEGF-C (Cys156Ser) protein (E-VEGF-C) and delivered it orally in different models of rat cirrhosis to target mLVs. Cirrhotic rats were given nanoformulation without VEGF-C served as vehicles. Drainage of mLVs was analysed using tracer dye. Portal and systemic physiological assessments and computed tomography were performed to measure portal pressures and ascites. Gene expression and permeability of primary mesenteric lymphatic endothelial cells (LyECs) was studied. Immune cells in mesenteric lymph nodes (MLNs) were quantified by flow cytometry. Endogenous and exogenous gut bacterial translocation to MLNs was examined. In cirrhotic rats, mLVs were dilated and leaky with impaired drainage. Treatment with E-VEGF-C induced proliferation of mLVs, reduced their diameter, and improved functional drainage. Ascites and portal pressures were significantly reduced in E-VEGF-C rats compared with vehicle rats. In MLNs of E-VEGF-C animals, CD8+CD134+ T cells were increased, whereas CD25+ regulatory T cells were decreased. Both endogenous and exogenous bacterial translocation were limited to MLNs in E-VEGF-C rats with reduced levels of endotoxins in ascites and blood in comparison with those in vehicle rats. E-VEGF-C treatment upregulated the expression of vascular endothelial-cadherin in LyECs and functionally improved the permeability of these cells. E-VEGF-C treatment ameliorates mesenteric lymph drainage and portal pressure and strengthens cytotoxic T-cell immunity in MLNs in experimental cirrhosis. It may thus serve as a promising therapy to manage ascites and reduce pathogenic gut bacterial translocation in cirrhosis. A human recombinant pro-lymphangiogenic growth factor, VEGF-C, was encapsulated in nanolipocarriers (E-VEGF-C) and orally delivered in different models of rat liver cirrhosis to facilitate its gut lymphatic vessel uptake. E-VEGF-C administration significantly increased mesenteric lymphatic vesselproliferation and improved lymph drainage, attenuating abdominal ascites and portal pressures in the animal models. E-VEGF-C treatment limited bacterial translocation to MLNs only with reduced gut bacterial load and ascitic endotoxins. E-VEGF-C therapy thus holds the potential to manage ascites and portal pressure and reduce gut bacterial translocation in patients with cirrhosis.

Supraphysiological levels of testosterone induces endothelial injury via increasing generation of reactive oxygen species and ERK1/2 activation

Introduction: High levels of testosterone (Testo) is associated with higher cardiovascular risk by positively modulate the generation of reactive oxygen species (ROS) and inflammatory response. Excessive ROS generation by NADPH oxidases (NOX, major source of ROS in the vasculature) leads to endothelial dysfunction, apoptosis, and inflammation. However, the intracellular mechanisms whereby supraphysiological levels of Testo promote cardiovascular damage are still ill defined. We sought to determine whether supraphysiological levels of Testo induces endothelial cell dysfunction via ROS generation and ERK1/2 activation. Methods: To study whether Testo present the same effects independent on endothelial cell type, we used Primary Human Mesenteric vascular Endothelial Cells (HMEC) and Human Umbilical Vein Endothelial Cells (HUVEC). Cells were stimulated with Testo (10 −7 M, for different time points) in the presence or absence of superoxide dismutase mimetic (Tempol, 10 -4 M, for 30 min). NOX activity, gene expression, and protein expression were determined pharmacological assays and immunoblotting, respectively. Results: Testo increased superoxide anion (O 2 − ) generation in HMEC and HUVEC at short (10 min) stimulation times, which was abolished by superoxide dismutase (SOD) mimetic [(% of control) Ctrl: 100.0 ± 0.0 n=5 vs Testo: 363.2 ± 19.77 n=6 vs. Tempol+Testo: 156.7 ± 12.12 n=6, *P<0.05]. Furthermore, long-term treatment (24 h) of endothelial cells with Testo increased Nox4 expression at gene (2.44-Fold change) and protein levels [(% of control) Ctrl: 100.0 ± 0.0 n=4 vs. Testo: 157.3 ± 4.49 n=4, *P<0.05]. Testo did not affect Nox1, Nox2, and Nox5 expressions. Furthermore, Testo increased ERK1/2 phosphorylation at 10 minutes and promoted endothelial cell inflammation at 8h, characterized by increased ICAM-1(6.49-Fold changes) and VCAM-1 [(1.42-Fold changes) gene expressions. To confirm that Testo-induced ROS production is leading to endothelial injury via ERK1/2, we treated endothelial cells with Tempol prior Testo treatment. Interestingly, superoxide dismutase mimetic prevented ERK1/2 phosphorylation induced by Testo [(% of control) Testo: 161.2 ± 11.39 n=5 vs. Tempol+Testo: 91.45 ± 9.61 n=5, *P<0.05]. Conclusion: These data indicate that supraphysiological levels of testosterone induce endothelial dysfunction via ROS generation and inflammatory response. Our data helps understanding the intracellular mechanisms that justify the cardiovascular damage caused by supraphysiological doses of testosterone and places antioxidant therapy as a possible pharmacological approach to treat patients with dysregulated testosterone levels. 2022/00103-3, São Paulo Research Foundation (FAPESP). NHLBI-R00 (R00HL14013903), AHA-CDA (CDA857268), Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania Vitalant, and Children's Hospital of Pittsburgh of the UPMC Health System. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heterogeneous membrane cholesterol in large and small rat mesenteric endothelial cells

Introduction: Endothelial dysfunction is a common event in cardiovascular disease, characterized by damage to the endothelium and impaired local blood flow regulation, including the regulation by the gasotransmitters, nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H 2 S). We and others have shown that H 2 S is a vasodilator; however, not all arterial segments respond similarly to this gasotransmitter. We have shown H 2 S-sensitivity in large arteries (>200 μm) is dependent on the membrane cholesterol content of endothelial cells (EC), while smaller arteries (<200 μm) are inherently highly sensitive to H 2 S. In addition, we have demonstrated ECs from large arteries contain more cholesterol in the plasma membrane than ECs from smaller arteries. Taken together, these results suggest that EC signaling is dependent on the level of membrane cholesterol and that the cholesterol content of the plasma membrane is differentially regulated in arterial segments within the same organ. This study investigated the mechanism mediating differences in endothelial membrane cholesterol between large and small arteries. Our single-cell RNAseq data suggests that the differences in cholesterol content between small and large arteries may result from increased EC expression of ATP-binding cassette family a1 (Abca1) in small arteries. Therefore, we hypothesized that augmented cholesterol efflux by Abca1 mediates the lower membrane cholesterol in ECs of resistance arteries facilitating vasodilatory signaling pathways. Methods: Endothelial tubes were generated by enzymatic digestion followed by titration of mesenteric arteries from male Sprague-Dawley rats (Envigo 175 – 225g, N=5 for all experiments). Cholesterol in endothelial tubes was measured using filipin-lll (50 μg/ml), while Abca1 was measured using immunofluorescence staining and confocal microscopy. We performed in-vitro studies manipulating membrane cholesterol by treating rat mesenteric ECs with Abca1 inhibitor, probucol (10μM), or Abca1 knockdown (KD) through RNA interference. Results: Our data reveal an increase in filipin-lll fluorescence in large artery ECs compared to ECs from small arteries and that Abca1 expression is greater in small artery ECs. Inhibition and KD of Abca1 using probucol or siRNA, respectively, ­ in vitro increased EC membrane cholesterol. Conclusions: Abca1 is an important regulator of EC membrane cholesterol and mediates the lower EC membrane cholesterol in small arteries compared to large arteries. Future investigations will explore whether hypercholesterolemia causes an increase in EC membrane cholesterol in the microcirculation resulting in endothelial dysfunction and impaired organ blood flow control. Funding: 1) NIH 1R01HL160606 (JSN), 2) T32HL007736 (JRA), and 3) UNM Comprehensive Cancer Center Support Grant NCI P30CA118100 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelial KCa3.1 and KCa2.3 Mediate S1P (Sphingosine-1-Phosphate)-Dependent Vasodilation and Blood Pressure Homeostasis.

S1P (sphingosine-1-phosphate) has been reported to possess vasodilatory properties, but the underlying pathways are largely unknown. Isolated mouse mesenteric artery and endothelial cell models were used to determine S1P-induced vasodilation, intracellular calcium, membrane potentials, and calcium-activated potassium channels (KCa2.3 and KCa3.1 [endothelial small- and intermediate-conductance calcium-activated potassium channels]). Effect of deletion of endothelial S1PR1 (type 1 S1P receptor) on vasodilation and blood pressure was evaluated. Mesenteric arteries subjected to acute S1P stimulation displayed a dose-dependent vasodilation response, which was attenuated by blocking endothelial KCa2.3 or KCa3.1 channels. In cultured human umbilical vein endothelial cells, S1P stimulated immediate membrane potential hyperpolarization following activation of KCa2.3/KCa3.1 with elevated cytosolic Ca2+. Further, chronic S1P stimulation enhanced expression of KCa2.3 and KCa3.1 in human umbilical vein endothelial cells in dose- and time-dependent manners, which was abolished by disrupting either S1PR1-Ca2+ signaling or downstream Ca2+-activated calcineurin/NFAT (nuclear factor of activated T-cells) signaling. By combination of bioinformatics-based binding site prediction and chromatin immunoprecipitation assay, we revealed in human umbilical vein endothelial cells that chronic activation of S1P/S1PR1 promoted NFATc2 nuclear translocation and binding to promoter regions of KCa2.3 and KCa3.1 genes thus to upregulate transcription of these channels. Deletion of endothelial S1PR1 reduced expression of KCa2.3 and KCa3.1 in mesenteric arteries and exacerbated hypertension in mice with angiotensin II infusion. This study provides evidence for the mechanistic role of KCa2.3/KCa3.1-activated endothelium-dependent hyperpolarization in vasodilation and blood pressure homeostasis in response to S1P. This mechanistic demonstration would facilitate the development of new therapies for cardiovascular diseases associated with hypertension.

Acid-sensing ion channel 1a activates IKCa/SKCa channels and contributes to endothelium-dependent dilation.

Acid-sensing ion channel 1a (ASIC1a) belongs to a novel family of proton-gated cation channels that are permeable to both Na+ and Ca2+. ASIC1a is expressed in vascular smooth muscle and endothelial cells in a variety of vascular beds, yet little is known regarding the potential impact of ASIC1a to regulate local vascular reactivity. Our previous studies in rat mesenteric arteries suggest ASIC1a does not contribute to agonist-induced vasoconstriction but may mediate a vasodilatory response. The objective of the current study is to determine the role of ASIC1a in systemic vasodilatory responses by testing the hypothesis that the activation of endothelial ASIC1a mediates vasodilation of mesenteric resistance arteries through an endothelium-dependent hyperpolarization (EDH)-related pathway. The selective ASIC1a antagonist psalmotoxin 1 (PcTX1) largely attenuated the sustained vasodilatory response to acetylcholine (ACh) in isolated, pressurized mesenteric resistance arteries and ACh-mediated Ca2+ influx in freshly isolated mesenteric endothelial tubes. Similarly, basal tone was enhanced and ACh-induced vasodilation blunted in mesenteric arteries from Asic1a knockout mice. ASIC1a colocalizes with intermediate- and small-conductance Ca2+-activated K+ channels (IKCa and SKCa, respectively), and the IKCa/SKCa-sensitive component of the ACh-mediated vasodilation was blocked by ASIC1a inhibition. To determine the role of ASIC1a to activate IKCa/SKCa channels, we measured whole-cell K+ currents using the perforated-patch clamp technique in freshly isolated mesenteric endothelial cells. Inhibition of ASIC1a prevented ACh-induced activation of IKCa/SKCa channels. The ASIC1 agonist, α/β-MitTx, activated IKCa/SKCa channels and induced an IKCa/SKCa-dependent vasodilation. Together, the present study demonstrates that ASIC1a couples to IKCa/SKCa channels in mesenteric resistance arteries to mediate endothelium-dependent vasodilation.

Ligustilide Prevents Radiation Enteritis by Targeting Gch1/BH4/eNOS to Improve Intestinal Ischemia.

There is a high incidence of radiation enteritis (RE) after abdominal radiotherapy. The occurrence of RE seriously affects the treatment and quality of life of patients; however, its pathogenesis is complex and there are no effective drugs for its prevention or treatment. Intestinal ischemia plays an important role in the occurrence of enteritis. Previous studies have shown that targeting GTP-cyclohydrolase 1 (Gch1) to improve intestinal ischemia could be a new strategy to prevent and treat RE. A high content of the naturally occurring phthalide derivative ligustilide (LIG) has been found in the plant drug Rhizoma Ligustici Chuanxiong for the treatment of cardiovascular diseases. The purpose of this study was to evaluate the protective effects of LIG on RE. Ionizing radiation (IR) rat and endothelial cell models were used to observe and record rat body weights and stool morphologies, measure intestinal blood perfusion by laser Doppler blood flow imaging, determine the diastolic functions of mesenteric arteries, detect the levels of Gch1/BH4/eNOS pathway-related proteins and regulatory molecules in the mesenteric arteries and endothelial cells, and predict affinity by molecular docking technology. The results showed that LIG significantly improved the body weights, loose stools, intestinal villi lengths, intestinal perfusion and vasodilatory functions of IR rats. LIG also significantly improved Gch1 protein and BH4 levels in the mesenteric arteries and endothelial cells after IR, increased the NO content, reduced superoxide accumulation, and improved p-eNOS (Ser1177) levels in endothelial cells. LIG has good affinity for Gch1, which significantly improves its activity. These results indicate that LIG is the preferred compound for the prevention and treatment of RE by improving intestinal ischemia through the Gch1/BH4/eNOS pathway. This study provides a theoretical basis and new research ideas for the development of new drugs for RE.

Novel Pannexin-1-Coupled Signaling Cascade Involved in the Control of Endothelial Cell Function and NO-Dependent Relaxation.

Deletion of pannexin-1 (Panx-1) leads not only to a reduction in endothelium-derived hyperpolarization but also to an increase in NO-mediated vasodilation. Therefore, we evaluated the participation of Panx-1-formed channels in the control of membrane potential and [Ca2+]i of endothelial cells. Changes in NO-mediated vasodilation, membrane potential, superoxide anion (O2·–) formation, and endothelial cell [Ca2+]i were analyzed in rat isolated mesenteric arterial beds and primary cultures of mesenteric endothelial cells. Inhibition of Panx-1 channels with probenecid (1 mM) or the Panx-1 blocking peptide 10Panx (60 μM) evoked an increase in the ACh (100 nM)-induced vasodilation of KCl-contracted mesenteries and in the phosphorylation level of endothelial NO synthase (eNOS) at serine 1177 (P-eNOSS1177) and Akt at serine 473 (P-AktS473). In addition, probenecid or 10Panx application activated a rapid, tetrodotoxin (TTX, 300 nM)-sensitive, membrane potential depolarization and [Ca2+]i increase in endothelial cells. Interestingly, the endothelial cell depolarization was converted into a transient spike after removing Ca2+ ions from the buffer solution and in the presence of 100 μM mibefradil or 10 μM Ni2+. As expected, Ni2+ also abolished the increment in [Ca2+]i. Expression of Nav1.2, Nav1.6, and Cav3.2 isoforms of voltage-dependent Na+ and Ca2+ channels was confirmed by immunocytochemistry. Furthermore, the Panx-1 channel blockade was associated with an increase in O2·– production. Treatment with 10 μM TEMPOL or 100 μM apocynin prevented the increase in O2·– formation, ACh-induced vasodilation, P-eNOSS1177, and P-AktS473 observed in response to Panx-1 inhibition. These findings indicate that the Panx-1 channel blockade triggers a novel complex signaling pathway initiated by the sequential activation of TTX-sensitive Nav channels and Cav3.2 channels, leading to an increase in NO-mediated vasodilation through a NADPH oxidase-dependent P-eNOSS1177, which suggests that Panx-1 may be involved in the endothelium-dependent control of arterial blood pressure.

KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells

Background and Purpose: Arterial diameter is dictated by the contractile state of the vascular smooth muscle cells (VSMCs), which is modulated by direct and indirect inputs from endothelial cells (ECs). Modulators of KCNQ-encoded kV7 channels have considerable impact on arterial diameter and these channels are known to be expressed in VSMCs but not yet defined in ECs. However, expression of kV7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of KV7 channels within rat mesenteric artery ECs.Experimental Approach: In rat mesenteric artery, KCNQ transcript and KV7 channel protein expression were determined via RT-qPCR, immunocytochemistry, immunohistochemistry and immunoelectron microscopy. Wire myography was used to determine vascular reactivity.Key Results: KCNQ transcript was identified in isolated ECs and VSMCs. KV7.1, KV7.4 and KV7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different KV7.2-5 activators S-1 and ML213. KIR2 blockers ML133, and BaCl2 also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. KV7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP.Conclusion and Implications: In rat mesenteric artery ECs, KV7.4 and KV7.5 channels are expressed, functionally interact with endothelial KIR2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies KV7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium of these vessels.

Ionizing radiation induces BH4 deficiency by downregulating GTP-cyclohydrolase 1, a novel target for preventing and treating radiation enteritis

Radiation enteritis (RE) is a common side effect after radiotherapy for abdominal cancer. RE pathogenesis is complicated, with no drugs available for prevention or treatments. Intestinal ischemia is a key factor in the occurrence and development of enteritis. The effect of ionizing radiation (IR) on intestinal ischemia is unknown. Deficiency of tetrahydrobiopterin (BH4) produced by GTP-cyclohydrolase 1 (Gch1) is important in ischemic diseases. This study focused on the relationship of Gch1/BH4 between intestinal ischemia in radiation enteritis. BH4 levels were analyzed by high-performance liquid chromatography in humans and rats after radiotherapy. Intestinal blood perfusion was measured by laser doppler flow imaging. Vascular ring tests determined the diastolic functions of rat mesenteric arteries. Gene, protein, and immunohistochemical staining experiments and inhibitor interventions were used to investigate Gch1 and endothelial NOS (eNOS) in rat mesenteric arteries and endothelial cells. The results showed that IR decreased BH4 levels in patients and rats after radiotherapy and decreased intestinal blood perfusion in rats. The degree of change in intestinal ischemia was consistent with intestinal villus injury. Gch1 mRNA and protein levels and nitric oxide (NO) production significantly decreased, while eNOS uncoupling in arterial and vascular endothelial cells strongly increased. BH4 supplementation improved eNOS uncoupling and NO levels in vascular endothelia after IR. The results of this study showed that downregulation of Gch1 in intestinal blood vessels after IR is an important target in RE. BH4 supplementation may prevent intestinal ischemia and improve vascular endothelial function after IR. These findings have clinical significance for the prevention and treatment of RE.

Increased Muscle Mass Restores a Healthy Vascular Endothelial Cell Phenotype in Obesity

ObjectiveInvestigate the effects of hypermuscularity on expression of genes vital to healthy endothelial function in a model of obesity‐driven cardiovascular disease.Methodsdb/db mice, a well characterized model of human obesity, were crossed with mice lacking myostatin, a myokine that inhibits muscle growth, to generate lean, obese, and hypermuscular obese mice. Using a column separation technique, fresh endothelial cells were isolated from the aortic and mesenteric arteries and mRNA expression of genes vital to endothelial health were assessed by qPCR.ResultsThe current study found that obesity significantly (p<0.05) increases expression of endothelial nitric oxide synthase (eNOS) and NADPH oxidase 1 (NOX1) by 15 to 25‐fold in both aortic and mesenteric endothelial cells in comparison to lean controls. Interestingly, expression of galectin‐3 (Gal‐3) in obesity increased significantly by 50‐fold in aortic endothelial cells, but 150‐fold in mesenteric endothelial cells. However, in endothelial cells from hypermuscular obese mice, overexpression of both NOX1 and Gal‐3 was ameliorated in both the aortic and mesenteric endothelium. These data indicate that hypermuscularity beneficially alters Gal‐3 overexpression, thereby decreasing injurious effects of NOX1 overexpression and restoring the endothelium to a favorable balance of eNOS and NOX1.ConclusionHypermuscularity improves endothelial NOS/NOX balance by decreasing levels of galectin‐3, an upstream contributor of NOX1 expression. Inhibition of galectin‐3 may prove to be a viable therapeutic target to ameliorate obesity‐driven cardiovascular disease.