Inflammation and DKK1-induced AKT activation contribute to endothelial dysfunction following NR2F2 loss.

NR2F2 is expressed in endothelial cells (ECs) and Nr2f2 knockout produces lethal cardiovascular defects. In humans, reduced NR2F2 expression is associated with cardiovascular diseases including congenital heart disease and atherosclerosis. Here, NR2F2 silencing in human primary ECs led to inflammation, endothelial-to-mesenchymal transition (EndMT), proliferation, hypermigration, apoptosis-resistance, and increased production of reactive oxygen species. These changes were associated with STAT and AKT activation along with increased production of DKK1. Co-silencing DKK1 and NR2F2 prevented NR2F2-loss-induced STAT and AKT activation and reversed EndMT. Serum DKK1 concentrations were elevated in patients with pulmonary arterial hypertension (PAH) and DKK1 was secreted by ECs in response to in vitro loss of either BMPR2 or CAV1, which are genetic defects associated with the development of PAH. In human primary ECs, NR2F2 suppressed DKK1, whereas its loss conversely induced DKK1 and disrupted endothelial homeostasis, promoting phenotypic abnormalities associated with pathologic vascular remodeling. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating chronic vascular diseases associated with EC dysfunction.NEW & NOTEWORTHY NR2F2 loss in the endothelial lining of blood vessels is associated with cardiovascular disease. Here, NR2F2-silenced human endothelial cells were inflammatory, proliferative, hypermigratory, and apoptosis-resistant with increased oxidant stress and endothelial-to-mesenchymal transition. DKK1 was induced in NR2F2-silenced endothelial cells, while co-silencing NR2F2 and DKK1 prevented NR2F2-loss-associated abnormalities in endothelial signaling and phenotype. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating vascular diseases associated with endothelial dysfunction.

Vaccination Ameliorates Cellular Inflammatory Responses in SARS-CoV-2 Breakthrough Infections.

BACKGROUND: Data on cellular immune responses in persons with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection following vaccination are limited. The evaluation of these patients with SARS-CoV-2 breakthrough infections may provide insight into how vaccinations limit the escalation of deleterious host inflammatory responses. METHODS: We conducted a prospective study of peripheral blood cellular immune responses to SARS-CoV-2 infection in 21 vaccinated patients, all with mild disease, and 97 unvaccinated patients stratified based on disease severity. RESULTS: We enrolled 118 persons (aged 50 years [SD 14.5 years], 52 women) with SARS-CoV-2 infection. Compared to unvaccinated patients, vaccinated patients with breakthrough infections had a higher percentage of antigen-presenting monocytes (HLA-DR+), mature monocytes (CD83+), functionally competent T cells (CD127+), and mature neutrophils (CD10+); and lower percentages of activated T cells (CD38+), activated neutrophils (CD64+), and immature B cells (CD127+CD19+). These differences widened with increased disease severity in unvaccinated patients. Longitudinal analysis showed that cellular activation decreased over time but persisted in unvaccinated patients with mild disease at 8-month follow-up. CONCLUSIONS: Patients with SARS-CoV-2 breakthrough infections exhibit cellular immune responses that limit the progression of inflammatory responses and suggest mechanisms by which vaccination limits disease severity. These data may have implications for developing more effective vaccines and therapies. Clinical Trials Registration. NCT04401449.

Mineralocorticoid receptor antagonist treatment of established pulmonary arterial hypertension improves interventricular dependence in the SU5416-hypoxia rat model.

Treatment with mineralocorticoid receptor (MR) antagonists beginning at the outset of disease, or early thereafter, prevents pulmonary vascular remodeling in preclinical models of pulmonary arterial hypertension (PAH). However, the efficacy of MR blockade in established disease, a more clinically relevant condition, remains unknown. Therefore, we investigated the effectiveness of two MR antagonists, eplerenone (EPL) and spironolactone (SPL), after the development of severe right ventricular (RV) dysfunction in the rat SU5416-hypoxia (SuHx) PAH model. Cardiac magnetic resonance imaging (MRI) in SuHx rats at the end of week 5, before study treatment, confirmed features of established disease including reduced RV ejection fraction and RV hypertrophy, pronounced septal flattening with impaired left ventricular filling and reduced cardiac index. Five weeks of treatment with either EPL or SPL improved left ventricular filling and prevented the further decline in cardiac index compared with placebo. Interventricular septal displacement was reduced by EPL whereas SPL effects were similar, but not significant. Although MR antagonists did not significantly reduce pulmonary artery pressure or vessel remodeling in SuHx rats with established disease, animals with higher drug levels had lower pulmonary pressures. Consistent with effects on cardiac function, EPL treatment tended to suppress MR and proinflammatory gene induction in the RV. In conclusion, MR antagonist treatment led to modest, but consistent beneficial effects on interventricular dependence after the onset of significant RV dysfunction in the SuHx PAH model. These results suggest that measures of RV structure and/or function may be useful endpoints in clinical trials of MR antagonists in patients with PAH.

The Role of Semaphorins and Their Receptors in Innate Immune Responses and Clinical Diseases of Acute Inflammation.

Semaphorins are a group of proteins that have been studied extensively for their critical function in neuronal development. They have been shown to regulate airway development, tumorigenesis, autoimmune diseases, and the adaptive immune response. Notably, emerging literature describes the role of immunoregulatory semaphorins and their receptors, plexins and neuropilins, as modulators of innate immunity and diseases defined by acute injury to the kidneys, abdomen, heart and lungs. In this review we discuss the pathogenic functions of semaphorins in clinical conditions of acute inflammation, including sepsis and acute lung injury, with a focus on regulation of the innate immune response as well as potential future therapeutic targeting.

Type I interferon activation and endothelial dysfunction in caveolin-1 insufficiency-associated pulmonary arterial hypertension.

Interferonopathies, interferon (IFN)-α/ß therapy, and caveolin-1 (CAV1) loss-of-function have all been associated with pulmonary arterial hypertension (PAH). Here, CAV1-silenced primary human pulmonary artery endothelial cells (PAECs) were proliferative and hypermigratory, with reduced cytoskeletal stress fibers. Signal transducers and activators of transcription (STAT) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) were both constitutively activated in these cells, resulting in a type I IFN-biased inflammatory signature. Cav1-/- mice that spontaneously develop pulmonary hypertension were found to have STAT1 and AKT activation in lung homogenates and increased circulating levels of CXCL10, a hallmark of IFN-mediated inflammation. PAH patients with CAV1 mutations also had elevated serum CXCL10 levels and their fibroblasts mirrored phenotypic and molecular features of CAV1-deficient PAECs. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 frameshift mutations. While blocking JAK/STAT or AKT rescued aspects of CAV1 loss, only AKT inhibitors suppressed activation of both signaling pathways simultaneously. Silencing endothelial nitric oxide synthase (NOS3) prevented STAT1 and AKT activation induced by CAV1 loss, implicating CAV1/NOS3 uncoupling and NOS3 dysregulation in the inflammatory phenotype. Exogenous IFN reduced CAV1 expression, activated STAT1 and AKT, and altered the cytoskeleton of PAECs, implicating these mechanisms in PAH associated with autoimmune and autoinflammatory diseases, as well as IFN therapy. CAV1 insufficiency elicits an IFN inflammatory response that results in a dysfunctional endothelial cell phenotype and targeting this pathway may reduce pathologic vascular remodeling in PAH.

Sphingosine-1-phosphate is involved in the occlusive arteriopathy of pulmonary arterial hypertension.

Despite several advances in the pathobiology of pulmonary arterial hypertension (PAH), its pathogenesis is not completely understood. Current therapy improves symptoms but has disappointing effects on survival. Sphingosine-1-phosphate (S1P) is a lysophospholipid synthesized by sphingosine kinase 1 (SphK1) and SphK2. Considering the regulatory roles of S1P in several tissues leading to vasoconstriction, inflammation, proliferation, and fibrosis, we investigated whether S1P plays a role in the pathogenesis of PAH. To test this hypothesis, we used plasma samples and lung tissue from patients with idiopathic PAH (IPAH) and the Sugen5416/hypoxia/normoxia rat model of occlusive PAH. Our study revealed an increase in the plasma concentration of S1P in patients with IPAH and in early and late stages of PAH in rats. We observed increased expression of both SphK1 and SphK2 in the remodeled pulmonary arteries of patients with IPAH and PAH rats. Exogenous S1P stimulated the proliferation of cultured rat pulmonary arterial endothelial and smooth-muscle cells. We also found that 3 weeks of treatment of late-stage PAH rats with an SphK1 inhibitor reduced the increased plasma levels of S1P and the occlusive pulmonary arteriopathy. Although inhibition of SphK1 improved cardiac index and the total pulmonary artery resistance index, it did not reduce right ventricular systolic pressure or right ventricular hypertrophy. Our study supports that S1P is involved in the pathogenesis of occlusive arteriopathy in PAH and provides further evidence that S1P signaling may be a novel therapeutic target.

MicroRNA-140 is elevated and mitofusin-1 is downregulated in the right ventricle of the Sugen5416/hypoxia/normoxia model of pulmonary arterial hypertension.

Heart failure, a major cause of morbidity and mortality in patients with pulmonary arterial hypertension (PAH), is an outcome of complex biochemical processes. In this study, we determined changes in microRNAs (miRs) in the right and left ventricles of normal and PAH rats. Using an unbiased quantitative miR microarray analysis, we found 1) miR-21-5p, miR-31-5 and 3p, miR-140-5 and 3p, miR-208b-3p, miR-221-3p, miR-222-3p, miR-702-3p, and miR-1298 were upregulated (>2-fold; P < 0.05) in the right ventricle (RV) of PAH compared with normal rats; 2) miR-31-5 and 3p, and miR-208b-3p were upregulated (>2-fold; P < 0.05) in the left ventricle plus septum (LV+S) of PAH compared with normal rats; 3) miR-187-5p, miR-208a-3p, and miR-877 were downregulated (>2-fold; P < 0.05) in the RV of PAH compared with normal rats; and 4) no miRs were up- or downregulated with >2-fold in LV+S compared with RV of PAH and normal. Upregulation of miR-140 and miR-31 in the hypertrophic RV was further confirmed by quantitative PCR. Interestingly, compared with control rats, expression of mitofusin-1 (MFN1), a mitochondrial fusion protein that regulates apoptosis, and which is a direct target of miR-140, was reduced in the RV relative to LV+S of PAH rats. We found a correlation between increased miR-140 and decreased MFN1 expression in the hypertrophic RV. Our results also demonstrated that upregulation of miR-140 and downregulation of MFN1 correlated with increased RV systolic pressure and hypertrophy. These results suggest that miR-140 and MFN1 play a role in the pathogenesis of PAH-associated RV dysfunction.

Raf/ERK drives the proliferative and invasive phenotype of BMPR2-silenced pulmonary artery endothelial cells.

A proliferative endothelial cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Bone morphogenetic protein type II receptor (BMPR2) loss-of-function is the most common cause of heritable PAH and has been closely linked to the formation of pathological plexiform lesions. Although some BMPR2 mutations leave ligand-dependent responses intact, the disruption of ligand-independent, noncanonical functions are universal among PAH-associated BMPR2 genotypes, but incompletely understood. This study examined the noncanonical signaling consequences of BMPR2 silencing in human pulmonary artery endothelial cells to identify potential therapeutic targets. BMPR2 siRNA silencing resulted in a proliferative, promigratory pulmonary artery endothelial cell phenotype and disruption of cytoskeletal architecture. Expression profiling closely reflected these phenotypic changes. Gene set enrichment and promoter analyses, as well as the differential expression of pathway components identified Ras/Raf/ERK signaling as an important consequence of BMPR2 silencing. Raf family members and ERK1/2 were constitutively activated after BMPR2 knockdown. Two Raf inhibitors, sorafenib and AZ628, and low-dose nintedanib, a triple receptor tyrosine kinase inhibitor upstream from Ras, reversed the abnormal proliferation and hypermotility of BMPR2 deficiency. Inhibition of dysregulated Ras/Raf/ERK signaling may be useful in reversing vascular remodeling in PAH.

Temporal hemodynamic and histological progression in Sugen5416/hypoxia/normoxia-exposed pulmonary arterial hypertensive rats.

We have investigated the temporal relationship between the hemodynamic and histological/morphological progression in a rat model of pulmonary arterial hypertension that develops pulmonary arterial lesions morphologically indistinguishable from those in human pulmonary arterial hypertension. Adult male rats were injected with Sugen5416 and exposed to hypoxia for 3 wk followed by a return to normoxia for various additional weeks. At 1, 3, 5, 8, and 13 wk after the Sugen5416 injection, hemodynamic and histological examinations were performed. Right ventricular systolic pressure reached its maximum 5 wk after Sugen5416 injection and plateaued thereafter. Cardiac index decreased at the 3∼5-wk time point, and tended to further decline at later time points. Reflecting these changes, calculated total pulmonary resistance showed a pattern of progressive worsening. Acute intravenous fasudil markedly reduced the elevated pressure and resistance at all time points tested. The percentage of severely occluded small pulmonary arteries showed a similar pattern of progression to that of right ventricular systolic pressure. These small vessels were occluded predominantly with nonplexiform-type neointimal formation except for the 13-wk time point. There was no severe occlusion in larger arteries until the 13-wk time point, when significant numbers of vessels were occluded with plexiform-type neointima. The Sugen5416/hypoxia/normoxia-exposed rat shows a pattern of chronic hemodynamic progression similar to that observed in pulmonary arterial hypertension patients. In addition to vasoconstriction, nonplexiform-type neointimal occlusion of small arteries appears to contribute significantly to the early phase of pulmonary arterial hypertension development, and plexiform-type larger vessel occlusion may play a role in the late deterioration.

Serotonin passes through myoendothelial gap junctions to promote pulmonary arterial smooth muscle cell differentiation.

Myoendothelial gap junctional signaling mediates pulmonary arterial endothelial cell (PAEC)-induced activation of latent TGF-ß and differentiation of cocultured pulmonary arterial smooth muscle cells (PASMCs), but the nature of the signal passing from PAECs to PASMCs through the gap junctions is unknown. Because PAECs but not PASMCs synthesize serotonin, and serotonin can pass through gap junctions, we hypothesized that the monoamine is the intercellular signal. We aimed to determine whether PAEC-derived serotonin mediates PAEC-induced myoendothelial gap junction-dependent activation of TGF-ß signaling and differentiation of PASMCs. Rat PAECs and PASMCs were monocultured or cocultured with (touch) or without (no-touch) direct cell-cell contact. In all cases, tryptophan hydroxylase 1 (Tph1) transcripts were expressed predominantly in PAECs. Serotonin was detected by immunostaining in both PAECs and PASMCs in PAEC/PASMC touch coculture but was not found in PASMCs in either PAEC/PASMC no-touch coculture or in PASMC/PASMC touch coculture. Furthermore, inhibition of gap junctions but not of the serotonin transporter in PAEC/PASMC touch coculture prevented serotonin transfer from PAECs to PASMCs. Inhibition of serotonin synthesis pharmacologically or by small interfering RNAs to Tph1 in PAECs inhibited the PAEC-induced activation of TGF-ß signaling and differentiation of PASMCs. We concluded that serotonin synthesized by PAECs is transferred through myoendothelial gap junctions into PASMCs, where it activates TGF-ß signaling and induces a more differentiated phenotype. This finding suggests a novel role of gap junction-mediated intercellular serotonin signaling in regulation of PASMC phenotype.

Myoendothelial gap junctional signaling induces differentiation of pulmonary arterial smooth muscle cells.

Myoendothelial gap junctions are involved in regulating systemic arterial smooth muscle cell phenotype and function, but their role in the regulation of pulmonary arterial smooth muscle cell (PASMC) phenotype is unknown. We therefore investigated in cocultured pulmonary arterial endothelial cells (PAECs) and PASMCs whether myoendothelial gap junctional signaling played a role in PAEC-dependent regulation of PASMC phenotype. Rat PAECs and PASMCs were cocultured on opposite sides of a porous Transwell membrane that permitted formation of heterotypic cell-cell contacts. Immunostaining showed expression of the gap junctional protein connexin 43 (Cx43) on projections extending into the membrane from both cell types. Dye transfer exhibited functional gap junctional communication from PAECs to PASMCs. PASMCs cocultured with PAECs had a more contractile-like phenotype (spindle shape and increased expression of the contractile proteins myosin heavy chain, H1-calponin, and α-smooth muscle cell-actin) than PASMCs cocultured with PASMCs or cocultured without direct contact with PAECs. Transforming growth factor (TGF)-ß1 signaling was activated in PASMCs cocultured with PAECs, and the PASMC differentiation was inhibited by TGF-ß type I receptor blockade. Inhibition of gap junctional communication pharmacologically or by knock down of Cx43 in PAECs blocked TGF-ß signaling and PASMC differentiation. These results implicate myoendothelial gap junctions as a gateway for PAEC-derived signals required for maintaining TGF-ß-dependent PASMC differentiation. This study identifies an alternative pathway to paracrine signaling to convey regulatory signals from PAECs to PASMCs and raises the possibility that dysregulation of this direct interaction is involved in the pathogenesis of hypertensive pulmonary vascular remodeling.

Tyrosine kinase inhibitors are potent acute pulmonary vasodilators in rats.

Tyrosine kinase inhibitors are promising for the treatment of severe pulmonary hypertension. Their therapeutic effects are postulated to be due to inhibition of cell growth-related kinases and attenuation of vascular remodeling. Their potential vasodilatory activities have not been explored. Vasorelaxant effects of the tyrosine kinase inhibitors imatinib, sorafenib, and nilotinib were examined in isolated pulmonary arterial rings from normal and pulmonary hypertensive rats. Phosphorylation of myosin light chain phosphatase and myosin light chain was assessed by Western blots. Acute hemodynamic effects of imatinib were tested in the pulmonary hypertensive rats. In normal pulmonary arteries, imatinib reversed serotonin- and U46619-induced contractions in a concentration-dependent and endothelium-independent manner. Sorafenib and nilotinib relaxed U46619-induced contraction. Imatinib inhibited activation of myosin phosphatase induced by U46619 in normal pulmonary arteries. All three tyrosine kinase inhibitors concentration-dependently and completely reversed the spontaneous contraction of hypertensive pulmonary arterial rings unmasked by inhibition of nitric oxide synthase. Acute intravenous administration of imatinib reduced high right ventricular systolic pressure in pulmonary hypertensive rats, with little effect on left ventricular systolic pressure and cardiac output. We conclude that tyrosine kinase inhibitors have potent pulmonary vasodilatory activity, which could contribute to their long-term beneficial effect against pulmonary hypertension. Vascular smooth muscle relaxation mediated via activation of myosin light chain phosphatase (Ca(2+) desensitization) appears to play a role in the imatinib-induced pulmonary vasodilation.

Reconstitution of the olfactory epithelium following injury in apoE-deficient mice.

ApoE, a protein component of lipoproteins, is extensively expressed in the primary olfactory pathway. Because apoE has been shown to play a vital role in nerve repair and remodeling, we hypothesized that apoE expression will increase in the injured olfactory epithelium (OE), and that apoE deficiency in apoE knockout (KO) mice will lead to delayed/incomplete reconstitution of the OE following injury. To directly test this hypothesis, we compared OE regeneration in wild-type (WT) and KO mice following injury induced by intranasal irrigation of Triton X-100. OE was collected at 0, 3, 7, 21, 42, and 56 days post lesion. The amount and distribution of apoE in the regenerating OE was measured by immunoblotting and immunohistochemistry. Rate of OE reconstitution in WT and KO mice was assessed by using three independent measures: (1) OE thickness was measured in cresyl-violet stained sections, (2) basal cell proliferation was determined by using bromodeoxyuridine (BrdU) staining, and (3) differentiation and maturation of olfactory sensory neurons were measured by immunoblotting and immunohistochemical analysis of growth associated protein (GAP) 43 and olfactory marker protein (OMP). The results revealed that apoE expression in the OE is highly regulated during the entire course of OE reconstitution post injury, and that apoE deficiency in apoE KO mice leads to delayed recovery of mature OMP(+) cells in the reconstituting OE. The data suggest that apoE production increases in the injured OE to facilitate maturation of olfactory sensory neurons.

Impact of apoE deficiency during synaptic remodeling in the mouse olfactory bulb.

In this study we examined the role of apoE on the rate of synaptic recovery in the olfactory bulb (OB) following olfactory epithelium (OE) lesioning in mice. We used both immunoblotting and immunohistochemical techniques to compare the density of OB synaptophysin (Syn, a synaptic marker) in apoE-gene deficient/knockout (KO) mice and wild-type (WT) mice following OE lesion. We found that the whole bulb concentrations of Syn, measured by immunoblotting, declined sharply following injury in both WT and KO mice during the degenerative phase (3-7 days). After this initial decline, the Syn concentration gradually increased to normal levels by 56 days in WT mice. In contrast, Syn concentration in KO mice did not recover by day 56 when Syn density in WT was essentially normal. Glomerular Syn density, measured by immunohistochemistry, found a lower density in KO mice at all time points post-lesion. This lower concentration of whole bulb Syn parallels the slower recovery of glomerular area in KO mice. The data indicate that apoE deficiency in KO mice is associated with a delayed recovery of the glomerular area and a slower recovery in Syn concentration in the OB.

The distribution of apolipoprotein E in mouse olfactory epithelium.

Previous studies from our laboratory suggest that apolipoprotein (apoE), a lipid transporting protein, facilitates olfactory nerve regeneration. We have shown that apoE is enriched in the olfactory nerve and around the glomeruli of the olfactory bulb (OB). The studies reported herein were undertaken to identify possible sources of apoE in the olfactory epithelium (OE). Immunoblotting results revealed apoE expression in the OE of wild-type (WT) mice, but not in apoE deficient/knockout (KO) mice. Immunohistochemical studies revealed that the perikarya and processes of sustentacular (Sus) cells expressed apoE-like immunoreactivity. Minimal neuronal apoE immunostaining was seen, although apoE was observed in the interstial spaces between olfactory receptor neurons (ORN). Substantial apoE-like immunoreactivity was localized to the endfeet and terminal process of Sus cells surrounding the basal cells. Double labeling immunocytochemical studies confirmed that the cell bodies and endfeet of Sus cells expressed high levels of apoE. The endothelial cells of blood vessels were intensely stained for apoE in the lamina propria. Cells forming Bowman's gland also immunostained for apoE. The apoE staining in the nerve fascicles was less intense, but was uniformly distributed throughout the core of the nerve bundles. Heavily stained cells, probably ensheathing glia, surrounded the nerve fascicles. These results revealed that apoE is expressed in the adult OE and lamina propria at strategic locations where it could facilitate the differentiation, maturation and axonal growth of the ORN, perhaps by recycling lipids from degenerating ORN for use by growing axons.