Transient receptor potential vanilloid 4 channel inhibition attenuates lung ischemia-reperfusion injury in a porcine lung transplant model.

OBJECTIVE: Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel important in many physiological and pathophysiological processes, including pulmonary disease. Using a murine model, we previously demonstrated that TRPV4 mediates lung ischemia-reperfusion injury, the major cause of primary graft dysfunction after transplant. The current study tests the hypothesis that treatment with a TRPV4 inhibitor will attenuate lung ischemia-reperfusion injury in a clinically relevant porcine lung transplant model. METHODS: A porcine left-lung transplant model was used. Animals were randomized to 2 treatment groups (n = 5/group): vehicle or GSK2193874 (selective TRPV4 inhibitor). Donor lungs underwent 30 minutes of warm ischemia and 24 hours of cold preservation before left lung allotransplantation and 4 hours of reperfusion. Vehicle or GSK2193874 (1 mg/kg) was administered to the recipient as a systemic infusion after recipient lung explant. Lung function, injury, and inflammatory biomarkers were compared. RESULTS: After transplant, left lung oxygenation was significantly improved in the TRPV4 inhibitor group after 3 and 4 hours of reperfusion. Lung histology scores and edema were significantly improved, and neutrophil infiltration was significantly reduced in the TRPV4 inhibitor group. TRPV4 inhibitor-treated recipients had significantly reduced expression of interleukin-8, high mobility group box 1, P-selectin, and tight junction proteins (occludin, claudin-5, and zonula occludens-1) in bronchoalveolar lavage fluid as well as reduced angiopoietin-2 in plasma, all indicative of preservation of endothelial barrier function. CONCLUSIONS: Treatment of lung transplant recipients with TRPV4 inhibitor significantly improves lung function and attenuates ischemia-reperfusion injury. Thus, selective TRPV4 inhibition may be a promising therapeutic strategy to prevent primary graft dysfunction after transplant.

The endothelium: gatekeeper to lung ischemia-reperfusion injury.

The success of lung transplantation is limited by the high rate of primary graft dysfunction due to ischemia-reperfusion injury (IRI). Lung IRI is characterized by a robust inflammatory response, lung dysfunction, endothelial barrier disruption, oxidative stress, vascular permeability, edema, and neutrophil infiltration. These events are dependent on the health of the endothelium, which is a primary target of IRI that results in pulmonary endothelial barrier dysfunction. Over the past 10 years, research has focused more on the endothelium, which is beginning to unravel the multi-factorial pathogenesis and immunologic mechanisms underlying IRI. Many important proteins, receptors, and signaling pathways that are involved in the pathogenesis of endothelial dysfunction after IR are starting to be identified and targeted as prospective therapies for lung IRI. In this review, we highlight the more significant mediators of IRI-induced endothelial dysfunction discovered over the past decade including the extracellular glycocalyx, endothelial ion channels, purinergic receptors, kinases, and integrins. While there are no definitive clinical therapies currently available to prevent lung IRI, we will discuss potential clinical strategies for targeting the endothelium for the treatment or prevention of IRI. The accruing evidence on the essential role the endothelium plays in lung IRI suggests that promising endothelial-directed treatments may be approaching the clinic soon. The application of therapies targeting the pulmonary endothelium may help to halt this rapid and potentially fatal injury.

Pannexin-3 stabilizes the transcription factor Bcl6 in a channel-independent manner to protect against vascular oxidative stress.

Targeted degradation regulates the activity of the transcriptional repressor Bcl6 and its ability to suppress oxidative stress and inflammation. Here, we report that abundance of endothelial Bcl6 is determined by its interaction with Golgi-localized pannexin 3 (Panx3) and that Bcl6 transcriptional activity protects against vascular oxidative stress. Consistent with data from obese, hypertensive humans, mice with an endothelial cell-specific deficiency in Panx3 had spontaneous systemic hypertension without obvious changes in channel function, as assessed by Ca2+ handling, ATP amounts, or Golgi luminal pH. Panx3 bound to Bcl6, and its absence reduced Bcl6 protein abundance, suggesting that the interaction with Panx3 stabilized Bcl6 by preventing its degradation. Panx3 deficiency was associated with increased expression of the gene encoding the H2O2-producing enzyme Nox4, which is normally repressed by Bcl6, resulting in H2O2-induced oxidative damage in the vasculature. Catalase rescued impaired vasodilation in mice lacking endothelial Panx3. Administration of a newly developed peptide to inhibit the Panx3-Bcl6 interaction recapitulated the increase in Nox4 expression and in blood pressure seen in mice with endothelial Panx3 deficiency. Panx3-Bcl6-Nox4 dysregulation occurred in obesity-related hypertension, but not when hypertension was induced in the absence of obesity. Our findings provide insight into a channel-independent role of Panx3 wherein its interaction with Bcl6 determines vascular oxidative state, particularly under the adverse conditions of obesity.

Purinergic P2Y2 receptor-induced activation of endothelial TRPV4 channels mediates lung ischemia-reperfusion injury.

Lung ischemia-reperfusion injury (IRI), characterized by inflammation, vascular permeability, and lung edema, is the major cause of primary graft dysfunction after lung transplantation. Here, we investigated the cellular mechanisms underlying lung IR-induced activation of endothelial TRPV4 channels, which play a central role in lung edema and dysfunction after IR. In a left lung hilar-ligation model of IRI in mice, we found that lung IRI increased the efflux of ATP through pannexin 1 (Panx1) channels at the endothelial cell (EC) membrane. Elevated extracellular ATP activated Ca2+ influx through endothelial TRPV4 channels downstream of purinergic P2Y2 receptor (P2Y2R) signaling. P2Y2R-dependent activation of TRPV4 channels was also observed in human and mouse pulmonary microvascular endothelium in ex vivo and in vitro models of IR. Endothelium-specific deletion of P2Y2R, TRPV4, or Panx1 in mice substantially prevented lung IRI-induced activation of endothelial TRPV4 channels and lung edema, inflammation, and dysfunction. These results identify endothelial P2Y2R as a mediator of the pathological sequelae of IRI in the lung and show that disruption of the endothelial Panx1-P2Y2R-TRPV4 signaling pathway could be a promising therapeutic strategy for preventing lung IRI after transplantation.

Purinergic P2Y2 Receptor-Induced Activation of Endothelial TRPV4 Channels Mediates Lung Ischemia-Reperfusion Injury.

Lung ischemia-reperfusion injury (IRI), characterized by inflammation, vascular permeability, and lung edema, is the major cause of primary graft dysfunction after lung transplantation. We recently reported that endothelial cell (EC) TRPV4 channels play a central role in lung edema and dysfunction after IR. However, the cellular mechanisms for lung IR-induced activation of endothelial TRPV4 channels are unknown. In a left-lung hilar ligation model of IRI in mice, we found that lung IR increases the efflux of extracellular ATP (eATP) through pannexin 1 (Panx1) channels at the EC membrane. Elevated eATP activated elementary Ca2+ influx signals through endothelial TRPV4 channels through purinergic P2Y2 receptor (P2Y2R) signaling. P2Y2R-dependent activation of TRPV4 channels was also observed in human and mouse pulmonary microvascular endothelium in ex vivo and in vitro surrogate models of lung IR. Endothelium-specific deletion of P2Y2R, TRPV4, and Panx1 in mice had substantial protective effects against lung IR-induced activation of endothelial TRPV4 channels, lung edema, inflammation, and dysfunction. These results identify endothelial P2Y2R as a novel mediator of lung edema, inflammation, and dysfunction after IR, and show that disruption of endothelial Panx1-P2Y2R-TRPV4 signaling pathway could represent a promising therapeutic strategy for preventing lung IRI after transplantation.

The Polyanionic Drug Suramin Neutralizes Histones and Prevents Endotheliopathy.

Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but challenges with dosing or side effects such as bleeding limit clinical application. In this study, we demonstrate that suramin, a widely available polyanionic drug, completely neutralizes the toxic effects of individual histones, but not citrullinated histones from neutrophil extracellular traps. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sublethal doses of histones in vivo. Suramin also prevented histone-induced lung endothelial cell cytotoxicity in vitro and lung edema, intra-alveolar hemorrhage, and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels.

FHL5 Controls Vascular Disease-Associated Gene Programs in Smooth Muscle Cells.

BACKGROUND: Genome-wide association studies have identified hundreds of loci associated with common vascular diseases, such as coronary artery disease, myocardial infarction, and hypertension. However, the lack of mechanistic insights for many GWAS loci limits their translation into the clinic. Among these loci with unknown functions is UFL1-four-and-a-half LIM (LIN-11, Isl-1, MEC-3) domain 5 (FHL5; chr6q16.1), which reached genome-wide significance in a recent coronary artery disease/ myocardial infarction GWAS meta-analysis. UFL1-FHL5 is also associated with several vascular diseases, consistent with the widespread pleiotropy observed for GWAS loci. METHODS: We apply a multimodal approach leveraging statistical fine-mapping, epigenomic profiling, and ex vivo analysis of human coronary artery tissues to implicate FHL5 as the top candidate causal gene. We unravel the molecular mechanisms of the cross-phenotype genetic associations through in vitro functional analyses and epigenomic profiling experiments in coronary artery smooth muscle cells. RESULTS: We prioritized FHL5 as the top candidate causal gene at the UFL1-FHL5 locus through expression quantitative trait locus colocalization methods. FHL5 gene expression was enriched in the smooth muscle cells and pericyte population in human artery tissues with coexpression network analyses supporting a functional role in regulating smooth muscle cell contraction. Unexpectedly, under procalcifying conditions, FHL5 overexpression promoted vascular calcification and dysregulated processes related to extracellular matrix organization and calcium handling. Lastly, by mapping FHL5 binding sites and inferring FHL5 target gene function using artery tissue gene regulatory network analyses, we highlight regulatory interactions between FHL5 and downstream coronary artery disease/myocardial infarction loci, such as FOXL1 and FN1 that have roles in vascular remodeling. CONCLUSIONS: Taken together, these studies provide mechanistic insights into the pleiotropic genetic associations of UFL1-FHL5. We show that FHL5 mediates vascular disease risk through transcriptional regulation of downstream vascular remodeling gene programs. These transacting mechanisms may explain a portion of the heritable risk for complex vascular diseases.

Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1.

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 - an inwardly rectifying potassium channel that regulates membrane hyperpolarization - contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function.

In situ investigations of failure mechanisms of silica fibers from the venus flower basket (Euplectella Aspergillum).

The fibers of the deep-sea sponge Euplectella aspergillum exhibit exceptional mechanical properties due to their unique layered structure at a micrometer length scale. In the present study, we utilize a correlative approach comprising of in situ tensile testing inside a scanning electron microscope (SEM) and post-failure fractography to precisely understand mechanisms through which layered architecture of fibers fracture and improves damage tolerance in tensile loading condition. The real-time observation of fibers in the present study confirms for the first time that the failure starts from the surface of fibers and proceeds to the center through successive layers. The concentric layers surrounding the central core sacrifice themselves and protect the central core through various toughening mechanisms like crack deflection, crack arrest, interface debonding, and fiber pullout. STATEMENT OF SIGNIFICANCE: Biological materials often exhibit multiscale hierarchical structures that can be incorporated into the design of next generation of engineering materials. The fibers of deep-sea sponge E. aspergillum possess core-shell like layered architecture. Our in situ study reveals astounding strategies by which this architecture delays the fracture of the fiber. The core-shell architecture of these fibers behaves like fiber-reinforced ceramic matrix composite, where the outer shells act as a matrix and the central core acts as a fiber. The outer shells take the environmental brunt and scarify themselves to protect the central core. The precise understanding of damage evolution presented here will help to design architected materials for load-bearing applications.

New NSAID Conjugates as Potent and Selective COX-2 Inhibitors: Synthesis, Molecular Modeling and Biological Investigation.

New sets of ibuprofen and indomethacin conjugates comprising triazolyl heterocycle were synthesized via click chemistry, adopting an optimized protocol through the molecular hybridization approach affording the targeted agents in good yields. The new non-steroidal anti-inflammatory drug (NSAID) conjugates were designed and synthesized and could be considered as potential drug candidates for the treatment of pain and inflammation. The anti-inflammatory properties were investigated for all the synthesized conjugates. Among 14 synthesized conjugates, four (5a, 5b, 5d, and 5e) were found to have significant anti-inflammatory properties potency 117.6%, 116.5%, 93.8%, and 109.1% in comparison to reference drugs ibuprofen (97.2%) and indomethacin (100%) in the rat paw edema carrageenan test without any ulcerogenic liability. The suppression effect of cytokines IL-6, TNF-α, and iNOS in addition to NO in the LPS-induced RAW264.7 cells supports the promising anti-inflammatory properties observed in the ibuprofen conjugates. In addition, several conjugates showed promising peripheral and central analgesic activity. The selectivity index (SI) of compound 5a (23.096) indicates the significant efficacy and selectivity for COX-2 over COX-1. Molecular modeling (docking and QSAR) studies described the observed biological properties.

Endothelial IK and SK channel activation decreases pulmonary arterial pressure and vascular remodeling in pulmonary hypertension.

Endothelial cells (ECs) from small pulmonary arteries (PAs) release nitric oxide (NO) and prostacyclin, which lower pulmonary arterial pressure (PAP). In pulmonary hypertension (PH), the levels of endothelium-derived NO and prostacyclin are reduced, contributing to elevated PAP. Small-and intermediate-conductance Ca2+-activated K+ channels (IK and SK)-additional crucial endothelial mediators of vasodilation-are also present in small PAs, but their function has not been investigated in PH. We hypothesized that endothelial IK and SK channels can be targeted to lower PAP in PH. Whole-cell patch-clamp experiments showed functional IK and SK channels in ECs, but not smooth muscle cells, from small PAs. Using a SU5416 plus chronic hypoxia (Su + CH) mouse model of PH, we found that currents through EC IK and SK channels were unchanged compared with those from normal mice. Moreover, IK/SK channel-mediated dilation of small PAs was preserved in Su + CH mice. Consistent with previous reports, endothelial NO levels and NO-mediated dilation were reduced in small PAs from Su + CH mice. Notably, acute treatment with IK/SK channel activators decreased PAP in Su + CH mice but not in normal mice. Further, chronic activation of IK/SK channels decreased PA remodeling and right ventricular hypertrophy, which are pathological hallmarks of PH, in Su + CH mice. Collectively, our data provide the first evidence that, unlike endothelial NO release, IK/SK channel activity is not altered in PH. Our results also demonstrate proof of principle that IK/SK channel activation can be used as a strategy for lowering PAP in PH.

Endothelial TRPV4 channels in lung edema and injury.

The alveolo-capillary barrier is relatively impermeable, and facilitates gas exchange via the large alveolar surface in the lung. Disruption of alveolo-capillary barrier leads to accumulation of edema fluid in lung injury. Studies in animal models of various forms of lung injury provide evidence that TRPV4 channels play a critical role in disruption of the alveolo-capillary barrier and pathogenesis of lung injury. TRPV4 channels from capillary endothelial cells, alveolar epithelial cells, and immune cells have been implicated in the pathogenesis of lung injury. Recent studies in endothelium-specific TRPV4 knockout mice point to a central role for endothelial TRPV4 channels in lung injury. In this chapter, we review the findings on the pathological roles of endothelial TRPV4 channels in different forms of lung injury and future directions for further investigation.

Endothelial alpha globin is a nitrite reductase.

Resistance artery vasodilation in response to hypoxia is essential for matching tissue oxygen and demand. In hypoxia, erythrocytic hemoglobin tetramers produce nitric oxide through nitrite reduction. We hypothesized that the alpha subunit of hemoglobin expressed in endothelium also facilitates nitrite reduction proximal to smooth muscle. Here, we create two mouse strains to test this: an endothelial-specific alpha globin knockout (EC Hba1Δ/Δ) and another with an alpha globin allele mutated to prevent alpha globin's inhibitory interaction with endothelial nitric oxide synthase (Hba1WT/Δ36-39). The EC Hba1Δ/Δ mice had significantly decreased exercise capacity and intracellular nitrite consumption in hypoxic conditions, an effect absent in Hba1WT/Δ36-39 mice. Hypoxia-induced vasodilation is significantly decreased in arteries from EC Hba1Δ/Δ, but not Hba1WT/Δ36-39 mice. Hypoxia also does not lower blood pressure in EC Hba1Δ/Δ mice. We conclude the presence of alpha globin in resistance artery endothelium acts as a nitrite reductase providing local nitric oxide in response to hypoxia.

Novel Smooth Muscle Ca2+-Signaling Nanodomains in Blood Pressure Regulation.

BACKGROUND: Ca2+ signals in smooth muscle cells (SMCs) contribute to vascular resistance and control blood pressure. Increased vascular resistance in hypertension has been attributed to impaired SMC Ca2+ signaling mechanisms. In this regard, transient receptor potential vanilloid 4 (TRPV4SMC) ion channels are a crucial Ca2+ entry pathway in SMCs. However, their role in blood pressure regulation has not been identified. METHODS: We used SMC-specific TRPV4-/- (TRPV4SMC-/-) mice to assess the role of TRPV4SMC channels in blood pressure regulation. We determined the contribution of TRPV4SMC channels to the constrictor effect of α1 adrenergic receptor (α1AR) stimulation and elevated intraluminal pressure: 2 main physiologic stimuli that constrict resistance-sized arteries. The contribution of spatially separated TRPV4SMC channel subpopulations to elevated blood pressure in hypertension was evaluated in angiotensin II-infused mice and patients with hypertension. RESULTS: We provide first evidence that TRPV4SMC channel activity elevates resting blood pressure in normal mice. α1AR stimulation activated TRPV4SMC channels through PKCα (protein kinase Cα) signaling, which contributed significantly to vasoconstriction and blood pressure elevation. Intraluminal pressure-induced TRPV4SMC channel activity opposed vasoconstriction through activation of Ca2+-sensitive K+ (BK) channels, indicating functionally opposite pools of TRPV4SMC channels. Superresolution imaging of SMCs revealed spatially separated α1AR:TRPV4 and TRPV4:BK nanodomains in SMCs. These data suggest that spatially separated α1AR-TRPV4SMC and intraluminal pressure-TRPV4SMC-BK channel signaling have opposite effects on blood pressure, with α1AR-TRPV4SMC signaling dominating under resting conditions. Furthermore, in patients with hypertension and a mouse model of hypertension, constrictor α1AR-PKCα-TRPV4 signaling was upregulated, whereas dilator pressure-TRPV4-BK channel signaling was disrupted, thereby increasing vasoconstriction and elevating blood pressure. CONCLUSIONS: Our data identify novel smooth muscle Ca2+-signaling nanodomains that regulate blood pressure and demonstrate their impairment in hypertension.

Endothelial Transient Receptor Potential Vanilloid 4 Channels Mediate Lung Ischemia-Reperfusion Injury.

BACKGROUND: Lung ischemia-reperfusion injury (IRI), involving severe inflammation and edema, is a major cause of primary graft dysfunction after transplant. Activation of transient receptor potential vanilloid 4 (TRPV4) channels modulates vascular permeability. Thus, this study tests the hypothesis that endothelial TRPV4 channels mediate lung IRI. METHODS: A left lung hilar-ligation model was used to induce lung IR in C57BL/6 wild-type (WT), Trpv4-/-, tamoxifen-inducible endothelial Trpv4 knockout (Trpv4EC-/-), and tamoxifen-treated control (Trpv4fl/fl) (n ≥ 6 mice/group). WT mice were also treated with GSK2193874 (WT+GSK219), a TRPV4-specific inhibitor (1 mg/kg). Partial pressure of arterial oxygen, edema (wet-to-dry weight ratio), compliance, neutrophil infiltration, and cytokine concentrations in bronchoalveolar lavage fluid were assessed. Pulmonary microvascular endothelial cells were characterized in vitro after exposure to hypoxia-reoxygenation. RESULTS: Compared with WT, partial pressure of arterial oxygen after IR was significantly improved in Trpv4-/- mice (133.1 ± 43.9 vs 427.8 ± 83.1 mm Hg, P < .001) and WT+GSK219 mice (133.1 ± 43.9 vs 447.0 ± 67.6 mm Hg, P < .001). Pulmonary edema and neutrophil infiltration were also significantly reduced after IR in Trpv4-/- and WT+GSK219 mice vs WT. Trpv4EC-/- mice after IR demonstrated significantly improved oxygenation vs control (109.2 ± 21.6 vs 405.3 ± 41.4 mm Hg, P < .001) as well as significantly improved compliance and significantly less edema, neutrophil infiltration, and proinflammatory cytokine production (tumor necrosis factor-a, chemokine [C-X-C motif] ligand 1, interleukin 17, interferon-γ). Hypoxia-reoxygenation-induced permeability and chemokine (C-X-C motif) ligand 1 expression by pulmonary microvascular endothelial cells were significantly attenuated by TRPV4 inhibitors. CONCLUSIONS: Endothelial TRPV4 plays a key role in vascular permeability and lung inflammation after IR. TRPV4 channels may be a promising therapeutic target to mitigate lung IRI and decrease the incidence of primary graft dysfunction after transplant.

Endothelial pannexin 1-TRPV4 channel signaling lowers pulmonary arterial pressure in mice.

Pannexin 1 (Panx1), an ATP-efflux pathway, has been linked with inflammation in pulmonary capillaries. However, the physiological roles of endothelial Panx1 in the pulmonary vasculature are unknown. Endothelial transient receptor potential vanilloid 4 (TRPV4) channels lower pulmonary artery (PA) contractility and exogenous ATP activates endothelial TRPV4 channels. We hypothesized that endothelial Panx1-ATP-TRPV4 channel signaling promotes vasodilation and lowers pulmonary arterial pressure (PAP). Endothelial, but not smooth muscle, knockout of Panx1 increased PA contractility and raised PAP in mice. Flow/shear stress increased ATP efflux through endothelial Panx1 in PAs. Panx1-effluxed extracellular ATP signaled through purinergic P2Y2 receptor (P2Y2R) to activate protein kinase Cα (PKCα), which in turn activated endothelial TRPV4 channels. Finally, caveolin-1 provided a signaling scaffold for endothelial Panx1, P2Y2R, PKCα, and TRPV4 channels in PAs, promoting their spatial proximity and enabling signaling interactions. These results indicate that endothelial Panx1-P2Y2R-TRPV4 channel signaling, facilitated by caveolin-1, reduces PA contractility and lowers PAP in mice.