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The emergence of SARS-CoV-2 variants with mutations in the spike protein is raising concerns about the efficacy of infection- or vaccine-induced antibodies. We compared antibody binding and live virus neutralization of sera from naturally infected and Moderna-vaccinated individuals against two SARS-CoV-2 variants: B.1 containing the spike mutation D614G and the emerging B.1.351 variant containing additional spike mutations and deletions. Sera from acutely infected and convalescent COVID-19 patients exhibited a 3-fold reduction in binding antibody titers to the B.1.351 variant receptor-binding domain of the spike protein and a 3.5-fold reduction in neutralizing antibody titers against SARS-CoV-2 B.1.351 variant compared to the B.1 variant. Similar results were seen with sera from Moderna-vaccinated individuals. Despite reduced antibody titers against the B.1.351 variant, sera from infected and vaccinated individuals containing polyclonal antibodies to the spike protein could still neutralize SARS-CoV-2 B.1.351, suggesting that protective humoral immunity may be retained against this variant., Graphical abstract, In this study, Edara et al. (2021) report that, despite reduced antibody binding to the B.1.351 RBD, sera from infected (acute and convalescent) and Moderna (mRNA-1273)-vaccinated individuals were still able to neutralize the SARS-CoV-2 B.1.351 variant, suggesting that protective humoral immunity may be retained against this variant.

The emergence of SARS-CoV-2 variants with mutations in the spike protein is raising concerns about the efficacy of infection- or vaccine-induced antibodies. We compared antibody binding and live virus neutralization of sera from naturally infected and Moderna-vaccinated individuals against two SARS-CoV-2 variants: B.1 containing the spike mutation D614G and the emerging B.1.351 variant containing additional spike mutations and deletions. Sera from acutely infected and convalescent COVID-19 patients exhibited a 3-fold reduction in binding antibody titers to the B.1.351 variant receptor-binding domain of the spike protein and a 3.5-fold reduction in neutralizing antibody titers against SARS-CoV-2 B.1.351 variant compared to the B.1 variant. Similar results were seen with sera from Moderna-vaccinated individuals. Despite reduced antibody titers against the B.1.351 variant, sera from infected and vaccinated individuals containing polyclonal antibodies to the spike protein could still neutralize SARS-CoV-2 B.1.351, suggesting that protective humoral immunity may be retained against this variant.

(1) Background: The influenza virus continues to cause significant annual morbidity and mortality. The overall efficacy of seasonal influenza vaccination is suboptimal, which is partly due to host immune factors. The effects of imprinting and repeated seasonal influenza vaccination were investigated to assess for immune factors and mechanisms that impact influenza vaccine responses. (2) Methods: Twenty participants were enrolled into a prospective pilot study based on birth cohort and seasonal influenza immunization history. Immunologic parameters were assessed over a six-month period after the seasonal influenza vaccine was administered. (3) Results: There was no significant imprinting effect, as measured by hemagglutination inhibition (HAI) fold change, HAI geometric mean titer (GMT) for Day 29 or Day 180 post-vaccination and antigen- specific antibody-secreting cells (ASC) for Day 8 post-vaccination. Individuals who had minimal prior seasonal influenza vaccination had a higher magnitude ASC response and a higher HAI fold change post-vaccination than individuals who were repeatedly vaccinated. (4) Conclusions: Repeated seasonal influenza vaccination resulted in a decreased fold change of the immune response, although individuals in this cohort tended to have high HAI titers at baseline that persisted after vaccination. Imprinting effects were not observed in this cohort. These host immune factors should be considered in the development of universal influenza vaccines. ClinicalTrials.gov Identifier: NCT03686514

Pulmonary hypertension (PH) is a progressive disorder that causes significant morbidity and mortality despite existing therapies. PH pathogenesis is characterized by metabolic derangements that increase pulmonary artery smooth muscle cell (PASMC) proliferation and vascular remodeling. PH-associated decreases in peroxisome proliferator-activated receptor γ (PPARγ) stimulate PASMC proliferation, and PPARγ in coordination with PPARγ coactivator 1α (PGC1α) regulates mitochondrial gene expression and biogenesis. To further examine the impact of decreases in PPARγ expression on human PASMC (HPASMC) mitochondrial function, we hypothesized that depletion of either PPARγ or PGC1α perturbs mitochondrial structure and function to stimulate PASMC proliferation. To test this hypothesis, HPASMCs were exposed to hypoxia and treated pharmacologically with the PPARγ antagonist GW9662 or with siRNA against PPARγ or PGC1α for 72 hours. HPASMC proliferation (cell counting), target mRNA levels (qRT-PCR), target protein levels (Western blotting), mitochondria-derived H2O2 (confocal immunofluorescence), mitochondrial mass and fragmentation, and mitochondrial bioenergetic profiling were determined. Hypoxia or knockdown of either PPARγ or PGC1α increased HPASMC proliferation, enhanced mitochondria-derived H2O2, decreased mitochondrial mass, stimulated mitochondrial fragmentation, and impaired mitochondrial bioenergetics. Taken together, these findings provide novel evidence that loss of PPARγ diminishes PGC1α and stimulates derangements in mitochondrial structure and function that cause PASMC proliferation. Overexpression of PGC1α reversed hypoxia-induced HPASMC derangements. This study identifies additional mechanistic underpinnings of PH, and provides support for the notion of activating PPARγ as a novel therapeutic strategy in PH.

Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that signaling events involved in hypoxia-induced cell proliferation include sustained nuclear factor-kappaB (NF-κB) activation, increased NADPH oxidase 4 (Nox4) expression, and downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) levels. To further understand the role of reduced PPARγ levels associated with PH pathobiology, siRNA was employed to reduce PPARγ levels in human pulmonary artery smooth muscle cells (HPASMC) in vitro under normoxic conditions. PPARγ protein levels were reduced to levels comparable to those observed under hypoxic conditions. Depletion of PPARγ for 24-72 h activated mitogen-activated protein kinase, ERK 1/2, and NF-κB. Inhibition of ERK 1/2 prevented NF-κB activation caused by PPARγ depletion, indicating that ERK 1/2 lies upstream of NF-κB activation. Depletion of PPARγ for 72 h increased NF-κB-dependent Nox4 expression and H2O2 production. Inhibition of NF-κB or Nox4 attenuated PPARγ depletion-induced HPASMC proliferation. Degradation of PPARγ depletion-induced H2O2 by PEG-catalase prevented HPASMC proliferation and also ERK 1/2 and NF-κB activation and Nox4 expression, indicating that H2O2 participates in feed-forward activation of the above signaling events. Contrary to the effects of PPARγ depletion, HPASMC PPARγ overexpression reduced ERK 1/2 and NF-κB activation, Nox4 expression, and cell proliferation. Taken together these findings provide novel evidence that PPARγ plays a central role in the regulation of the ERK1/2-NF-κB-Nox4-H2O2 signaling axis in HPASMC. These results indicate that reductions in PPARγ caused by pathophysiological stimuli such as prolonged hypoxia exposure are sufficient to promote the proliferation of pulmonary vascular smooth muscle cells observed in PH pathobiology.

The ligand-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), regulates metabolism, cell proliferation, and inflammation. Pulmonary hypertension (PH) is associated with reduced PPARγ expression, and hypoxia exposure regimens that cause PH reduce PPARγ expression. The current study examines mechanisms of hypoxia-induced PPARγ downregulation in vitro and in vivo. Hypoxia reduced PPARγ mRNA and protein levels, PPARγ activity, and the expression of PPARγ regulated genes in human pulmonary artery smooth muscle cells (HPASMC) exposed to 1% oxygen for 72 hours. Similarly, exposure of mice to hypoxia (10% O2) for 3 weeks reduced PPARγ mRNA and protein in mouse lung. Inhibiting ERK1/2 with PD98059 or treatment with siRNA directed against either NF-κB p65 or Nox4 attenuated hypoxic reductions in PPARγ expression and activity. Furthermore, degradation of H2O2 using PEG-catalase prevented hypoxia-induced ERK 1/2 phosphorylation and Nox4 expression suggesting sustained ERK 1/2-mediated signaling and Nox4 expression in this response. Mammalian two hybrid assays demonstrated that PPARγ and p65 bind directly to each other in a mutually repressive fashion. Taken together, we conclude that hypoxic regimens that promote PH pathogenesis and HPASMC proliferation reduce PPARγ expression and activity through ERK1/2-, p65-, and Nox4-dependent pathways. These findings provide novel insights into mechanisms by which pathophysiological stimuli such as hypoxia cause loss of PPARγ activity and pulmonary vascular cell proliferation, pulmonary vascular remodeling, and PH. These results also indicate that restoration of PPARγ activity with pharmacological ligands may provide a novel therapeutic approach in selected forms of PH.

Peroxisome proliferator-activated receptor (PPAR) γ activation attenuates hypoxia-induced pulmonary hypertension (PH) in mice. The current study examined the hypothesis that PPARγ attenuates hypoxia-induced endothelin-1 (ET-1) signaling to mediate these therapeutic effects. To test this hypothesis, human pulmonary artery endothelial cells (HPAECs) were exposed to normoxia or hypoxia (1% O2) for 72 h and treated with or without the PPARγ ligand rosiglitazone (RSG, 10 μM) during the final 24 h of exposure. HPAEC proliferation was measured with MTT assays or cell counting, and mRNA and protein levels of ET-1 signaling components were determined. To explore the role of hypoxia-activated transcription factors, selected HPAECs were treated with inhibitors of hypoxia-inducible factor (HIF)-1α (chetomin) or nuclear factor (NF)-κB (caffeic acid phenethyl ester, CAPE). In parallel studies, male C57BL/6 mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 wk with or without gavage with RSG (10 mg·kg−1·day−1) for the final 10 days of exposure. Hypoxia increased ET-1, endothelin-converting enzyme-1, and endothelin receptor A and B levels in mouse lung and in HPAECs and increased HPAEC proliferation. Treatment with RSG attenuated hypoxia-induced activation of HIF-1α, NF-κB activation, and ET-1 signaling pathway components. Similarly, treatment with chetomin or CAPE prevented hypoxia-induced increases in HPAEC ET-1 mRNA and protein levels. These findings indicate that PPARγ activation attenuates a program of hypoxia-induced ET-1 signaling by inhibiting activation of hypoxia-responsive transcription factors. Targeting PPARγ represents a novel therapeutic strategy to inhibit enhanced ET-1 signaling in PH pathogenesis.

Vascular endothelial cells express the ligand-activated transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ), which participates in the regulation of metabolism, cell proliferation, and inflammation. PPARγ ligands attenuate, whereas the loss of function mutations in PPARγ stimulate, endothelial dysfunction, suggesting that PPARγ may regulate vascular endothelial nitric oxide production. To explore the role of endothelial PPARγ in the regulation of vascular nitric oxide production in vivo, mice expressing Cre recombinase driven by an endothelial-specific promoter were crossed with mice carrying a floxed PPARγ gene to produce endothelial PPARγ null mice (ePPARγ−/−). When compared with littermate controls, ePPARγ−/−animals were hypertensive at baseline and demonstrated comparable increases in systolic blood pressure in response to angiotensin II infusion. When compared with those of control animals, aortic ring relaxation responses to acetylcholine were impaired, whereas relaxation responses to sodium nitroprusside were unaffected in ePPARγ−/−mice. Similarly, intact aortic segments from ePPARγ−/−mice released less nitric oxide than those from controls, whereas endothelial nitric oxide synthase expression was similar in control and ePPARγ−/−aortas. Reduced nitric oxide production in ePPARγ−/−aortas was associated with an increase in the parameters of oxidative stress in the blood and the activation of nuclear factor-κB in aortic homogenates. These findings demonstrate that endothelial PPARγ regulates vascular nitric oxide production and that the disruption of endothelial PPARγ contributes to endothelial dysfunction in vivo.

Pathogenesis of pulmonary hypertension is complex and involves activation of the transcription factor, hypoxia-inducible factor-1 (HIF-1) that shifts cellular metabolism from aerobic respiration to glycolysis, in part, by increasing the expression of its downstream target pyruvate dehydrogenase kinase-1 (PDK-1), thereby promoting a proliferative, apoptosis-resistant phenotype in pulmonary vascular cells. Activation of the nuclear hormone transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), attenuates pulmonary hypertension and pulmonary artery smooth muscle cell (PASMC) proliferation. In the current study, we determined whether PPARγ inhibits HIF-1α and PDK-1 expression in human PASMCs.HPASMCs were exposed to normoxia (21% O2) or hypoxia (1% O2) for 2-72 hours ± treatment with the PPARγ-ligand, rosiglitazone (RSG, 10μM).Compared to normoxia, HIF-1α mRNA levels were elevated in HPASMC at 2 hours hypoxia and reduced to baseline levels by 24-72 hours. HIF-1α protein levels increased following 4 and 8 hours of hypoxia and returned to baseline levels by 24 and 72 hours. PDK-1 protein levels increased following 24 hours hypoxia and remained elevated by 72 hours. RSG treatment at the onset of hypoxia attenuated HIF-1α protein and PDK-1 mRNA and protein levels at 4, 8 and 24 hours of hypoxia, respectively. However, RSG treatment during final 24 hours of 72-hour hypoxia, an intervention that inhibits HPASMC proliferation, failed to prevent hypoxia-induced PDK-1 expression.Hypoxia causes transient activation of HPASMC HIF-1α that is attenuated by RSG treatment initiated at hypoxia onset. These findings provide novel evidence that PPARγ modulates fundamental and acute cellular responses to hypoxia through both HIF-1-dependent and HIF-1-independent mechanisms.

Endothelin-1 (ET-1) plays a critical role in endothelial dysfunction and contributes to the pathogenesis of pulmonary hypertension (PH). We hypothesized that peroxisome proliferator–activated receptor γ (PPARγ) stimulates microRNAs that inhibit ET-1 and pulmonary artery endothelial cell (PAEC) proliferation. The objective of this study was to clarify molecular mechanisms by which PPARγ regulates ET-1 expression in vitro and in vivo. In PAECs isolated from patients with pulmonary arterial hypertension, microRNA (miR)-98 expression was reduced, and ET-1 protein levels and proliferation were increased. Similarly, hypoxia reduced miR-98 and increased ET-1 levels and PAEC proliferation in vitro. In vivo, hypoxia reduced miR-98 expression and increased ET-1 and proliferating cell nuclear antigen (PCNA) levels in mouse lung, derangements that were aggravated by treatment with the vascular endothelial growth factor receptor antagonist Sugen5416. Reporter assays confirmed that miR-98 binds directly to the ET-1 3′-untranslated region. Compared with littermate control mice, miR-98 levels were reduced and ET-1 and PCNA expression were increased in lungs from endothelial-targeted PPARγ knockout mice, whereas miR-98 levels were increased and ET-1 and PCNA expression was reduced in lungs from endothelial-targeted PPARγ–overexpression mice. Gain or loss of PPARγ function in PAECs in vitro confirmed that alterations in PPARγ were sufficient to regulate miR-98, ET-1, and PCNA expression. Finally, PPARγ activation with rosiglitazone regimens that attenuated hypoxia-induced PH in vivo and human PAEC proliferation in vitro restored miR-98 levels. The results of this study show that PPARγ regulates miR-98 to modulate ET-1 expression and PAEC proliferation. These results further clarify molecular mechanisms by which PPARγ participates in PH pathogenesis and therapy.

Activation of the nuclear hormone receptor, PPARγ, with pharmacological agonists promotes a contractile vascular smooth muscle cell phenotype and reduces oxidative stress and cell proliferation, particularly under pathological conditions including vascular injury, restenosis, and atherosclerosis. However, pharmacological agonists activate both PPARγ-dependent and -independent mechanisms in multiple cell types confounding efforts to clarify the precise role of PPARγ in smooth muscle cell structure and function in vivo. We, therefore, designed and characterized a mouse model with smooth muscle cell-targeted PPARγ overexpression (smPPARγOE). Our results demonstrate that smPPARγOE attenuated contractile responses in aortic rings, increased aortic compliance, caused aortic dilatation, and reduced mean arterial pressure. Molecular characterization revealed that compared to littermate control mice, aortas from smPPARγOE mice expressed lower levels of contractile proteins and increased levels of adipocyte-specific transcripts. Morphological analysis demonstrated increased lipid deposition in the vascular media and in smooth muscle of extravascular tissues. In vitro adenoviral-mediated PPARγ overexpression in human aortic smooth muscle cells similarly increased adipocyte markers and lipid uptake. The findings demonstrate that smooth muscle PPARγ overexpression disrupts vascular wall structure and function, emphasizing that balanced PPARγ activity is essential for vascular smooth muscle homeostasis.

Rationale: Pulmonary hypertension (PH) is an important clinical problem that causes significant morbidity and mortality despite existing therapies. Recent evidence indicates that metabolic derangements in pulmonary vascular wall cells stimulate mitochondrial (MT) dysfunction that promotes proliferative vascular remodeling. We previously showed that expression of peroxisome proliferator-activated receptor gamma (PPARγ) was reduced in murine hypoxia-induced PH and that reductions in PPARγ were sufficient to stimulate Nox4 expression, H2O2 production, and human pulmonary artery smooth muscle cell (HPASMC) proliferation. Objective: We hypothesize that PPARγ coactivator 1 alpha (PGC1α, which stimulates MT biogenesis), is a critical PPARγ-regulated target that modulates MT function, reactive oxygen species (ROS) production, and the proliferative PASMC phenotype in PH. Methods and Results: To examine the molecular pathways stimulated by reductions in PPARγ, siPPARγ or GW9662 (PPARγ antagonist) were used to inhib...

Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that hypoxia stimulates sustained nuclear factor-kappaB (NF-κB) activation, increased NADPH oxidase4 (Nox4) expression, and downregulation of peroxisome proliferator-activated receptor-gamma (PPARγ) to cause human pulmonary artery smooth muscle cell (HPASMC) proliferation. To further understand the role of reduced PPARγ in PH pathobiology, siRNA was employed to reduce PPARγ levels in HPASMC in vitro under normoxic conditions. PPARγ protein levels were reduced to levels comparable to those observed under hypoxic conditions. Knockdown of PPARγ for 24-72 hours activated mitogen-activated protein kinase, ERK 1/2, and NF-κB. Inhibition of ERK 1/2 prevented NF-κB activation caused by PPARγ knockdown indicating that ERK 1/2 lies upstream of NF-κB activation. Knockdown of PPARγ for 72 hours increased NF-κB-dependent Nox4 protein expression and H2O2 production. Degradati...

Objective: To explore the role of PPARγ in vascular regulation, smooth muscle VP16-PPARγ-overexpressing mice were subjected to studies of systemic vascular function, biomechanical testing, histological analysis, and molecular characterization. Methods: Mice with a flox-controlled VP16-PPARγ gene were crossed with mice containing a tamoxifen-inducible Cre recombinase driven by a smooth muscle myosin heavy chain promoter. All mice received tamoxifen injections at 6 weeks of age. Experiments were performed at 10-16 weeks of age. Results: Compared to littermate controls, transgenic mice displayed: 1) aortic dilation (by histology, echocardiography, and microCT), 2) increased aortic compliance, 3) reduced blood pressure, 4) 80% reduction in aortic ring contractile forces in response to KCl or phenylephrine, 5) lipid accumulation in the aortic media (Oil Red O staining), and 6) reduction in aortic contractile markers and extra-cellular matrix components with increased expression of adipose markers and selected ...

The complex pathobiology of pulmonary hypertension (PH) involves reactive oxygen species (ROS) and increased pulmonary vascular resistance. NADPH oxidase 4 (Nox4) has been shown to be an important ...

Hypoxia stimulates pulmonary hypertension in part by increasing the proliferation of pulmonary vascular wall cells via sustained activation of MAP kinase ERK 1/2, NF-κB, and downregulation of PPARγ levels. However, the upstream signaling events that mediate NF-κB upregulation and PPARγ downregulation remain unknown. We examined the role of Proline-rich tyrosine kinase 2 (Pyk2) in hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (HPASMC). Exposure of HPASMC to hypoxia (1% O2) for 72 hrs resulted in the activation of Pyk2. siRNA-mediated Pyk2 depletion or its pharmacological inhibition attenuated hypoxia-induced HPASMC proliferation. Pyk2 inhibition attenuated hypoxia-induced: 1) ERK 1/2 activation, 2) NF-κB transcriptional activity and 3) reductions in PPARγ mRNA levels. Conversely, siRNA-mediated PPARγ depletion enhanced, whereas PPARγ overexpression attenuated HPASMC Pyk2 activation. Exposing C57BL/6 mice to 10% O2 for 3 weeks reduced lung PPARγ but increased Pyk2 levels compar...

Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs, often fatally. Vascular endothelial cells are both key mediators and targets of LPS-induced inflammatory responses. The nuclear hormone receptor peroxisome proliferator–activated receptor γ (PPARγ) exerts anti-inflammatory actions in various cells, but it is unknown whether it modulates inflammation through actions within endothelial cells. To determine whether PPARγ acts within endothelial cells to diminish endotoxemic lung inflammation and injury, we measured inflammatory responses and mediators in mice with endothelial-targeted deletion of PPARγ. Endothelial cell PPARγ (ePPARγ) knockout exacerbated LPS-induced pulmonary inflammation and injury as shown by several measures, including infiltration of inflammatory cells, edema, and production of reactive oxygen species and proinflammatory cytokines, along with upregulation of the LPS receptor TLR4 in lung tissue and increased activation of its downstream signaling pathways. In isolated LPS-stimulated endothelial cells in vitro, absence of PPARγ enhanced the production of numerous inflammatory markers. We hypothesized that the observed in vivo activity of the ligand-activated ePPARγ may arise, in part, from nitrated fatty acids (NFAs), a novel class of endogenous PPARγ ligands. Supporting this idea, we found that treating isolated endothelial cells with physiologically relevant concentrations of the endogenous NFA 10-nitro-oleate reduced LPS-induced expression of a wide range of inflammatory markers in the presence of PPARγ, but not in its absence, and also inhibited neutrophil mobility in a PPARγ-dependent manner. Our results demonstrate a key protective role of ePPARγ against endotoxemic injury and a potential ePPARγ-mediated anti-inflammatory role for NFAs.

Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) contribute to hypoxia-induced pulmonary hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, GKT137831, in hypoxia-exposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O(2)) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARγ, and transforming growth factor (TGF)β1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O(2)) for 3 weeks with or without GKT137831 treatment during the final 10 days of exposure. Lung PPARγ and TGF-β1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H(2)O(2) release, proliferation, and TGF-β1 expression and blunted reductions in PPARγ in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-β1 and reductions in PPARγ expression and attenuated RVH and pulmonary artery wall thickness but not increases in RVSP or muscularization of small arterioles. This study shows that Nox4 plays a critical role in modulating proliferative responses of pulmonary vascular wall cells. Targeting Nox4 with GKT137831 provides a novel strategy to attenuate hypoxia-induced alterations in pulmonary vascular wall cells that contribute to vascular remodeling and RVH, key features involved in PH pathogenesis.