Digital Spatial Profiling Identifies Distinct Molecular Signatures of Vascular Lesions in Pulmonary Arterial Hypertension.

RATIONALE: Idiopathic Pulmonary Arterial Hypertension (IPAH) is characterized by extensive pulmonary vascular remodeling due to plexiform and obliterative lesions, media hypertrophy, inflammatory cell infiltration, and alterations of the adventitia. OBJECTIVE: Test the hypothesis that microscopic IPAH vascular lesions express unique molecular profiles, which collectively are different from control pulmonary arteries. METHODS: We used digital spatial transcriptomics to profile the genome-wide differential transcriptomic signature of key pathological lesions (plexiform, obliterative, intima+media hypertrophy, and adventitia) in IPAH lungs (n= 11) and compared these data to the intima+media and adventitia of control pulmonary artery (n=5). RESULTS: We detected 8273 transcripts in the IPAH lesions and control lung pulmonary arteries. Plexiform lesions and IPAH adventitia exhibited the greatest number of differentially expressed genes when compared with intima-media hypertrophy and obliterative lesions. Plexiform lesions in IPAH showed enrichment for (i) genes associated with TGFß-signaling and (ii) mutated genes affecting the extracellular matrix and endothelial-mesenchymal transformation. Plexiform lesions and IPAH adventitia showed upregulation of genes involved in immune and interferon signaling, coagulation, and complement pathways. Cellular deconvolution indicated variability in the number of vascular and inflammatory cells between IPAH lesions, which underlies the differential transcript profiling. CONCLUSIONS: IPAH lesions express unique molecular transcript profiles enriched for pathways involving pathogenetic pathways, including genetic disease drivers, innate and acquired immunity, hypoxia sensing, and angiogenesis signaling. These data provide a rich molecular-structural framework in IPAH vascular lesions that inform novel biomarkers and therapeutic targets in this highly morbid disease.

Lactate produced by alveolar type II cells suppresses inflammatory alveolar macrophages in acute lung injury.

Alveolar inflammation is a hallmark of acute lung injury (ALI), and its clinical correlate is acute respiratory distress syndrome-and it is as a result of interactions between alveolar type II cells (ATII) and alveolar macrophages (AM). In the setting of acute injury, the microenvironment of the intra-alveolar space is determined in part by metabolites and cytokines and is known to shape the AM phenotype. In response to ALI, increased glycolysis is observed in AT II cells, mediated by the transcription factor hypoxia-inducible factor (HIF) 1α, which has been shown to decrease inflammation. We hypothesized that in acute lung injury, lactate, the end product of glycolysis, produced by ATII cells shifts AMs toward an anti-inflammatory phenotype, thus mitigating ALI. We found that local intratracheal delivery of lactate improved ALI in two different mouse models. Lactate shifted cytokine expression of murine AMs toward increased IL-10, while decreasing IL-1 and IL-6 expression. Mice with ATII-specific deletion of Hif1a and mice treated with an inhibitor of lactate dehydrogenase displayed exacerbated ALI and increased inflammation with decreased levels of lactate in the bronchoalveolar lavage fluid; however, all those parameters improved with intratracheal lactate. When exposed to LPS (to recapitulate an inflammatory stimulus as it occurs in ALI), human primary AMs co-cultured with alveolar epithelial cells had reduced inflammatory responses. Taken together, these studies reveal an innate protective pathway, in which lactate produced by ATII cells shifts AMs toward an anti-inflammatory phenotype and dampens excessive inflammation in ALI.

Ceramide as an endothelial cell surface receptor and a lung-specific lipid vascular target for circulating ligands.

The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.

SIRT3 Is a Critical Regulator of Mitochondrial Function of Fibroblasts in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a heterogeneous and life-threatening cardiopulmonary disorder in which mitochondrial dysfunction is believed to drive pathogenesis, although the underlying mechanisms remain unclear. To determine if abnormal SIRT3 (sirtuin 3) activity is related to mitochondrial dysfunction in adventitial fibroblasts from patients with idiopathic pulmonary arterial hypertension (IPAH) and hypoxic PH calves (PH-Fibs) and whether SIRT3 could be a potential therapeutic target to improve mitochondrial function, SIRT3 concentrations in control fibroblasts, PH-Fibs, and lung tissues were determined using quantitative real-time PCR and western blot. SIRT3 deacetylase activity in cells and lung tissues was determined using western blot, immunohistochemistry staining, and immunoprecipitation. Glycolysis and mitochondrial function in fibroblasts were measured using respiratory analysis and fluorescence-lifetime imaging microscopy. The effects of restoring SIRT3 activity (by overexpression of SIRT3 with plasmid, activation SIRT3 with honokiol, and supplementation with the SIRT3 cofactor nicotinamide adenine dinucleotide [NAD+]) on mitochondrial protein acetylation, mitochondrial function, cell proliferation, and gene expression in PH-Fibs were also investigated. We found that SIRT3 concentrations were decreased in PH-Fibs and PH lung tissues, and its cofactor, NAD+, was also decreased in PH-Fibs. Increased acetylation in overall mitochondrial proteins and SIRT3-specific targets (MPC1 [mitochondrial pyruvate carrier 1] and MnSOD2 [mitochondrial superoxide dismutase]), as well as decreased MnSOD2 activity, was identified in PH-Fibs and PH lung tissues. Normalization of SIRT3 activity, by increasing its expression with plasmid or with honokiol and supplementation with its cofactor NAD+, reduced mitochondrial protein acetylation, improved mitochondrial function, inhibited proliferation, and induced apoptosis in PH-Fibs. Thus, our study demonstrated that restoration of SIRT3 activity in PH-Fibs can reduce mitochondrial protein acetylation and restore mitochondrial function and PH-Fib phenotype in PH.

Repetitive schistosoma exposure causes perivascular lung fibrosis and persistent pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) can occur as a complication of schistosomiasis. In humans, schistosomiasis-PH persists despite antihelminthic therapy and parasite eradication. We hypothesized that persistent disease arises as a consequence of exposure repetition. METHODS: Following intraperitoneal sensitization, mice were experimentally exposed to Schistosoma eggs by intravenous injection, either once or three times repeatedly. The phenotype was characterized by right heart catheterization and tissue analysis. RESULTS: Following intraperitoneal sensitization, a single intravenous Schistosoma egg exposure resulted in a PH phenotype that peaked at 7-14 days, followed by spontaneous resolution. Three sequential exposures resulted in a persistent PH phenotype. Inflammatory cytokines were not significantly different between mice exposed to one or three egg doses, but there was an increase in perivascular fibrosis in those who received three egg doses. Significant perivascular fibrosis was also observed in autopsy specimens from patients who died of this condition. CONCLUSIONS: Repeatedly exposing mice to schistosomiasis causes a persistent PH phenotype, accompanied by perivascular fibrosis. Perivascular fibrosis may contribute to the persistent schistosomiasis-PH observed in humans with this disease.

The Effect of Hypoxia-inducible Factor Inhibition on the Phenotype of Fibroblasts in Human and Bovine Pulmonary Hypertension.

Hypoxia-inducible factor (HIF) has received much attention as a potential pulmonary hypertension (PH) treatment target because inhibition of HIF reduces the severity of established PH in rodent models. However, the limitations of small-animal models of PH in predicting the therapeutic effects of pharmacologic interventions in humans PH are well known. Therefore, we sought to interrogate the role of HIFs in driving the activated phenotype of PH cells from human and bovine vessels. We first established that pulmonary arteries (PAs) from human and bovine PH lungs exhibit markedly increased expression of direct HIF target genes (CA9, GLUT1, and NDRG1), as well as cytokines/chemokines (CCL2, CSF2, CXCL12, and IL6), growth factors (FGF1, FGF2, PDGFb, and TGFA), and apoptosis-resistance genes (BCL2, BCL2L1, and BIRC5). The expression of the genes found in the intact PAs was determined in endothelial cells, smooth muscle cells, and fibroblasts cultured from the PAs. The data showed that human and bovine pulmonary vascular fibroblasts from patients or animals with PH (termed PH-Fibs) were the cell type that exhibited the highest level and the most significant increases in the expression of cytokines/chemokines and growth factors. In addition, we found that human, but not bovine, PH-Fibs exhibit consistent misregulation of HIFα protein stability, reduced HIF1α protein hydroxylation, and increased expression of HIF target genes even in cells grown under normoxic conditions. However, whereas HIF inhibition reduced the expression of direct HIF target genes, it had no impact on other "persistently activated" genes. Thus, our study indicated that HIF inhibition alone is not sufficient to reverse the persistently activated phenotype of human and bovine PH-Fibs.

Integrative Multiomics to Dissect the Lung Transcriptional Landscape of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) remains an incurable and often fatal disease despite currently available therapies. Multiomics systems biology analysis can shed new light on PAH pathobiology and inform translational research efforts. Using RNA sequencing on the largest PAH lung biobank to date (96 disease and 52 control), we aim to identify gene co-expression network modules associated with PAH and potential therapeutic targets. Co-expression network analysis was performed to identify modules of co-expressed genes which were then assessed for and prioritized by importance in PAH, regulatory role, and therapeutic potential via integration with clinicopathologic data, human genome-wide association studies (GWAS) of PAH, lung Bayesian regulatory networks, single-cell RNA-sequencing data, and pharmacotranscriptomic profiles. We identified a co-expression module of 266 genes, called the pink module, which may be a response to the underlying disease process to counteract disease progression in PAH. This module was associated not only with PAH severity such as increased PVR and intimal thickness, but also with compensated PAH such as lower number of hospitalizations, WHO functional class and NT-proBNP. GWAS integration demonstrated the pink module is enriched for PAH-associated genetic variation in multiple cohorts. Regulatory network analysis revealed that BMPR2 regulates the main target of FDA-approved riociguat, GUCY1A2, in the pink module. Analysis of pathway enrichment and pink hub genes (i.e. ANTXR1 and SFRP4) suggests the pink module inhibits Wnt signaling and epithelial-mesenchymal transition. Cell type deconvolution showed the pink module correlates with higher vascular cell fractions (i.e. myofibroblasts). A pharmacotranscriptomic screen discovered ubiquitin-specific peptidases (USPs) as potential therapeutic targets to mimic the pink module signature. Our multiomics integrative study uncovered a novel gene subnetwork associated with clinicopathologic severity, genetic risk, specific vascular cell types, and new therapeutic targets in PAH. Future studies are warranted to investigate the role and therapeutic potential of the pink module and targeting USPs in PAH.

Tensions in Taxonomies: Current Understanding and Future Directions in the Pathobiologic Basis and Treatment of Group 1 and Group 3 Pulmonary Hypertension.

In the over 100 years since the recognition of pulmonary hypertension (PH), immense progress and significant achievements have been made with regard to understanding the pathophysiology of the disease and its treatment. These advances have been mostly in idiopathic pulmonary arterial hypertension (IPAH), which was classified as Group 1 Pulmonary Hypertension (PH) at the Second World Symposia on PH in 1998. However, the pathobiology of PH due to chronic lung disease, classified as Group 3 PH, remains poorly understood and its treatments thus remain limited. We review the history of the classification of the five groups of PH and aim to provide a state-of-the-art review of the understanding of the pathogenesis of Group 1 PH and Group 3 PH including insights gained from novel high-throughput omics technologies that have revealed heterogeneities within these categories as well as similarities between them. Leveraging the substantial gains made in understanding the genomics, epigenomics, proteomics, and metabolomics of PAH to understand the full spectrum of the complex, heterogeneous disease of PH is needed. Multimodal omics data as well as supervised and unbiased machine learning approaches after careful consideration of the powerful advantages as well as of the limitations and pitfalls of these technologies could lead to earlier diagnosis, more precise risk stratification, better predictions of disease response, new sub-phenotype groupings within types of PH, and identification of shared pathways between PAH and other types of PH that could lead to new treatment targets. © 2023 American Physiological Society. Compr Physiol 13:4295-4319, 2023.

HIF1A-dependent induction of alveolar epithelial PFKFB3 dampens acute lung injury.

Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us toward a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxP/loxP SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid instillation. Studies in genetic models linked Pfkfb3 expression and function to Hif1a. Not only did intratracheal pyruvate instillation reconstitute Pfkfb3loxP/loxP or Hif1aloxP/loxP SPC-ER-Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies demonstrated increased PFKFB3 staining in injured lungs and colocalized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counterbalancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI.

The role of macrophages in right ventricular remodeling in experimental pulmonary hypertension.

Right ventricular (RV) failure is the primary cause of death in pulmonary hypertension (PH), but the mechanisms of RV failure are not well understood. We hypothesized macrophages in the RV contribute to the RV response in PH. We induced PH in mice with hypoxia (FiO2 10%) and Schistosoma mansoni exposure, and in rats with SU5416-hypoxia. We quantified cardiac macrophages in mice using flow cytometry. Parabiosis between congenic CD45.1/.2 mice or Cx3cr1-green fluorescent protein and wild-type mice was used to quantify circulation-derived macrophages in experimental PH conditions. We administered clodronate liposomes to Sugen hypoxia (SU-Hx) exposed rats to deplete macrophages and evaluated the effect on the extracellular matrix (ECM) and capillary network in the RV. In hypoxia exposed mice, the overall number of macrophages did not significantly change but two macrophage subpopulations increased. Parabiosis identified populations of RV macrophages that at steady state is derived from the circulation, with one subpopulation that significantly increased with PH stimuli. Clodronate treatment of SU-Hx rats resulted in a change in the RV ECM, without altering the RV vasculature, and correlated with improved RV function. Populations of RV macrophages increase and contribute to RV remodeling in PH, including through regulation of the RV ECM.

Inhaled seralutinib exhibits potent efficacy in models of pulmonary arterial hypertension.

BACKGROUND: Signalling through platelet-derived growth factor receptor (PDGFR), colony-stimulating factor 1 receptor (CSF1R) and mast/stem cell growth factor receptor kit (c-KIT) plays a critical role in pulmonary arterial hypertension (PAH). We examined the preclinical efficacy of inhaled seralutinib, a unique small-molecule PDGFR/CSF1R/c-KIT kinase inhibitor in clinical development for PAH, in comparison to a proof-of-concept kinase inhibitor, imatinib. METHODS: Seralutinib and imatinib potency and selectivity were compared. Inhaled seralutinib pharmacokinetics/pharmacodynamics were studied in healthy rats. Efficacy was evaluated in two rat models of PAH: SU5416/Hypoxia (SU5416/H) and monocrotaline pneumonectomy (MCTPN). Effects on inflammatory/cytokine signalling were examined. PDGFR, CSF1R and c-KIT immunohistochemistry in rat and human PAH lung samples and microRNA (miRNA) analysis in the SU5416/H model were performed. RESULTS: Seralutinib potently inhibited PDGFRα/ß, CSF1R and c-KIT. Inhaled seralutinib demonstrated dose-dependent inhibition of lung PDGFR and c-KIT signalling and increased bone morphogenetic protein receptor type 2 (BMPR2). Seralutinib improved cardiopulmonary haemodynamic parameters and reduced small pulmonary artery muscularisation and right ventricle hypertrophy in both models. In the SU5416/H model, seralutinib improved cardiopulmonary haemodynamic parameters, restored lung BMPR2 protein levels and decreased N-terminal pro-brain natriuretic peptide (NT-proBNP), more than imatinib. Quantitative immunohistochemistry in human lung PAH samples demonstrated increased PDGFR, CSF1R and c-KIT. miRNA analysis revealed candidates that could mediate seralutinib effects on BMPR2. CONCLUSIONS: Inhaled seralutinib was an effective treatment of severe PAH in two animal models, with improved cardiopulmonary haemodynamic parameters, a reduction in NT-proBNP, reverse remodelling of pulmonary vascular pathology and improvement in inflammatory biomarkers. Seralutinib showed greater efficacy compared to imatinib in a preclinical study.

Contribution of fatty acid oxidation to the pathogenesis of pulmonary hypertension.

Dysregulated metabolism characterizes both animal and human forms of pulmonary hypertension (PH). Enzymes involved in fatty acid metabolism have previously not been assessed in human pulmonary arteries affected by pulmonary arterial hypertension (PAH), and how inhibition of fatty acid oxidation (FAO) may attenuate PH remains unclear. Fatty acid metabolism gene transcription was quantified in laser-dissected pulmonary arteries from 10 explanted lungs with advanced PAH (5 idiopathic, 5 associated with systemic sclerosis), and 5 donors without lung diseases. Effects of oxfenicine, a FAO inhibitor, on female Sugen 5416-chronic hypoxia (SuHx) rats were studied in vivo using right heart catheterization, and ex vivo using perfused lungs and pulmonary artery ring segments. The impact of pharmacologic (oxfenicine) and genetic (carnitine palmitoyltransferase 1a heterozygosity) FAO suppression was additionally probed in mouse models of Schistosoma and hypoxia-induced PH. Potential mechanisms underlying FAO-induced PH pathogenesis were examined by quantifying ATP and mitochondrial mass in oxfenicine-treated SuHx pulmonary arterial cells, and by assessing pulmonary arterial macrophage infiltration with immunohistochemistry. We found upregulated pulmonary arterial transcription of 26 and 13 FAO genes in idiopathic and systemic sclerosis-associated PAH, respectively. In addition to promoting de-remodeling of pulmonary arteries in SuHx rats, oxfenicine attenuated endothelin-1-induced vasoconstriction. FAO inhibition also conferred modest benefit in the two mouse models of PH. Oxfenicine increased mitochondrial mass in cultured rat pulmonary arterial cells, and decreased the density of perivascular macrophage infiltration in pulmonary arteries of treated SuHx rats. In summary, FAO inhibition attenuated experimental PH, and may be beneficial in human PAH.

Experimental Schistosoma japonicum-induced pulmonary hypertension.

BACKGROUND: Schistosomiasis, a major cause of pulmonary arterial hypertension (PAH) worldwide, is most clearly described complicating infection by one species, Schistosoma mansoni. Controlled exposure of mice can be used to induce Type 2 inflammation-dependent S. mansoni pulmonary hypertension (PH). We sought to determine if another common species, S. japonicum, can also cause experimental PH. METHODS: Schistosome eggs were obtained from infected mice, and administered by intraperitoneal sensitization followed by intravenous challenge to experimental mice, which underwent right heart catheterization and tissue analysis. RESULTS: S. japonicum sensitized and challenged mice developed PH, which was milder than that following S. mansoni sensitization and challenge. The degree of pulmonary vascular remodeling and Type 2 inflammation in the lungs was similarly proportionate. Cross-sensitization revealed that antigens from either species are sufficient to sensitize for intravenous challenge with either egg, and the degree of PH severity depended on primarily the species used for intravenous challenge. Compared to a relatively uniform distribution of S. mansoni eggs, S. japonicum eggs were observed in clusters in the lungs. CONCLUSIONS: S. japonicum can induce experimental PH, which is milder than that resulting from comparable S. mansoni exposure. This difference may result from the distribution of eggs in the lungs, and is independent of which species is used for sensitization. This result is consistent with the clearer association between S. mansoni infection and the development of schistosomiasis-associated PAH in humans.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

VEGF-C/VEGFR-3 signalling in macrophages ameliorates acute lung injury.

BACKGROUND: Successful recovery from acute lung injury requires inhibition of neutrophil influx and clearance of apoptotic neutrophils. However, the mechanisms underlying recovery remain unclear. We investigated the ameliorative effects of vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 (VEGFR-3) signalling in macrophages in lipopolysaccharide (LPS)-induced lung injury. METHODS: LPS was intranasally injected into wild-type and transgenic mice. Gain and loss of VEGF-C/VEGFR-3 signalling function experiments employed adenovirus-mediated intranasal delivery of VEGF-C (Ad-VEGF-C vector) and soluble VEGFR-3 (sVEGFR-3) or anti-VEGFR-3 blocking antibodies and mice with a deletion of VEGFR-3 in myeloid cells. RESULTS: The early phase of lung injury was significantly alleviated by the overexpression of VEGF-C with increased levels of bronchoalveolar lavage (BAL) fluid interleukin-10 (IL-10), but worsened in the later phase by VEGFR-3 inhibition upon administration of Ad-sVEGFR-3 vector. Injection of anti-VEGFR-3 antibodies to mice in the resolution phase inhibited recovery from lung injury. The VEGFR-3-deleted mice had a shorter survival time than littermates and more severe lung injury in the resolution phase. Alveolar macrophages in the resolution phase digested most of the extrinsic apoptotic neutrophils and VEGF-C/VEGFR-3 signalling increased efferocytosis via upregulation of integrin αv in the macrophages. We also found that incubation with BAL fluid from acute respiratory distress syndrome (ARDS) patients, but not from controls, decreased VEGFR-3 expression and the efficiency of IL-10 expression and efferocytosis in human monocyte-derived macrophages. CONCLUSIONS: VEGF-C/VEGFR-3 signalling in macrophages ameliorates experimental lung injury. This mechanism may also provide an explanation for ARDS resolution.

Phosphoproteomic analysis of lung tissue from patients with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare disorder associated with high morbidity and mortality despite currently available treatments. We compared the phosphoproteome of lung tissue from subjects with idiopathic PAH (iPAH) obtained at the time of lung transplant with control lung tissue. The mass spectrometry-based analysis found 60,428 phosphopeptide features from which 6622 proteins were identified. Within the subset of identified proteins there were 1234 phosphopeptides with q < 0.05, many of which are involved in immune regulation, angiogenesis, and cell proliferation. Most notably there was a marked relative increase in phosphorylated (S378) IKZF3 (Aiolos), a zinc finger transcription factor that plays a key role in lymphocyte regulation. In vitro phosphorylation assays indicated that GSK3 alpha and/or GSK3 beta could phosphorylate IKZF3 at S378. Western blot analysis demonstrated increased pIKZF3 in iPAH lungs compared to controls. Immunohistochemistry demonstrated phosphorylated IKZF3 in lymphocytes surrounding severely hypertrophied pulmonary arterioles. In situ hybrization showed gene expression in lymphocyte aggregates in PAH samples. A BCL2 reporter assay showed that IKZF3 increased BCL2 promoter activity and demonstrated the potential role of phosphorylation of IKZF3 in the regulation of BCL mediated transcription. Kinase network analysis demonstrated potentially important regulatory roles of casein kinase 2, cyclin-dependent kinase 1 (CDK1), mitogen-associated protein kinases (MAPKs), and protein kinases (PRKs) in iPAH. Bioinformatic analysis demonstrated enrichment of RhoGTPase signaling and the potential importance of cGMP-dependent protein kinase 1 (PRKG). In conclusion, this unbiased phosphoproteomic analysis demonstrated several novel targets regulated by kinase networks in iPAH, and reinforced the potential role of immune regulation in the pathogenesis of iPAH. The identified up- and down-regulated phosphoproteins have potential to serve as biomarkers for PAH and to provide new insights for therapeutic strategies.

Matrix-Degrading Enzyme Expression and Aortic Fibrosis During Continuous-Flow Left Ventricular Mechanical Support.

BACKGROUND: The effects of nonphysiological flow generated by continuous-flow (CF) left ventricular assist devices (LVADs) on the aorta remain poorly understood. OBJECTIVES: The authors sought to quantify indexes of fibrosis and determine the molecular signature of post-CF-LVAD vascular remodeling. METHODS: Paired aortic tissue was collected at CF-LVAD implant and subsequently at transplant from 22 patients. Aortic wall morphometry and fibrillar collagen content (a measure of fibrosis) was quantified. In addition, whole-transcriptome profiling by RNA sequencing and follow-up immunohistochemistry were performed to evaluate CF-LVAD-mediated changes in aortic mRNA and protein expression. RESULTS: The mean age was 52 ± 12 years, with a mean duration of CF-LVAD of 224 ± 193 days (range 45-798 days). There was a significant increase in the thickness of the collagen-rich adventitial layer from 218 ± 110 µm pre-LVAD to 410 ± 209 µm post-LVAD (P < 0.01). Furthermore, there was an increase in intimal and medial mean fibrillar collagen intensity from 22 ± 11 a.u. pre-LVAD to 41 ± 24 a.u. post-LVAD (P < 0.0001). The magnitude of this increase in fibrosis was greater among patients with longer durations of CF-LVAD support. CF-LVAD led to profound down-regulation in expression of extracellular matrix-degrading enzymes, such as matrix metalloproteinase-19 and ADAMTS4, whereas no evidence of fibroblast activation was noted. CONCLUSIONS: There is aortic remodeling and fibrosis after CF-LVAD that correlates with the duration of support. This fibrosis is due, at least in part, to suppression of extracellular matrix-degrading enzyme expression. Further research is needed to examine the contribution of nonphysiological flow patterns on vascular function and whether modulation of pulsatility may improve vascular remodeling and long-term outcomes.

Endothelial cell PHD2-HIF1α-PFKFB3 contributes to right ventricle vascular adaptation in pulmonary hypertension.

Humans and animals with pulmonary hypertension (PH) show right ventricular (RV) capillary growth, which positively correlates with overall RV hypertrophy. However, molecular drivers of RV vascular augmentation in PH are unknown. Prolyl hydroxylase (PHD2) is a regulator of hypoxia-inducible factors (HIFs), which transcriptionally activates several proangiogenic genes, including the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We hypothesized that a signaling axis of PHD2-HIF1α-PFKFB3 contributes to adaptive coupling between the RV vasculature and tissue volume to maintain appropriate vascular density in PH. We used design-based stereology to analyze endothelial cell (EC) proliferation and the absolute length of the vascular network in the RV free wall, relative to the tissue volume in mice challenged with hypoxic PH. We observed increased RV EC proliferation starting after 6 h of hypoxia challenge. Using parabiotic mice, we found no evidence for a contribution of circulating EC precursors to the RV vascular network. Mice with transgenic deletion or pharmacological inhibition of PHD2, HIF1α, or PFKFB3 all had evidence of impaired RV vascular adaptation following hypoxia PH challenge. PHD2-HIF1α-PFKFB3 contributes to structural coupling between the RV vascular length and tissue volume in hypoxic mice, consistent with homeostatic mechanisms that maintain appropriate vascular density. Activating this pathway could help augment the RV vasculature and preserve RV substrate delivery in PH, as an approach to promote RV function.

Targeted Phage Display-based Pulmonary Vaccination in Mice and Non-human Primates.

BACKGROUND: The extensive alveolar capillary network of the lungs is an attractive route for administration of several agents. One key functional attribute is the rapid onset of systemic action due to the absence of first-pass metabolism. METHODS: Here we applied a combinatorial approach for ligand-directed pulmonary delivery as a unique route for systemic targeting in vaccination. FINDINGS: We screened a phage display random peptide library in vivo to select, identify, and validate a ligand (CAKSMGDIVC) that specifically targets and is internalized through its receptor, α3ß1 integrin, on the surface of cells lining the lung airways and alveoli and mediates CAKSMGDIVC-displaying phage binding and systemic delivery without compromising lung homeostasis. As a proof-of-concept, we show that the pulmonary delivery of targeted CAKSMGDIVC-displaying phage particles in mice and non-human primates elicit a systemic and specific humoral response. CONCLUSIONS: This broad methodology blueprint represents a robust and versatile platform tool enabling new ligand-receptor discovery with many potential translational applications. FUNDING: Cancer Center Support Grants to the University of Texas M.D. Anderson Cancer Center (CA016672), University of New Mexico Comprehensive Cancer Center (CA118100), Rutgers Cancer Institute of New Jersey (CA072720), research awards from the Gillson Longenbaugh Foundation, and National Institutes of Health (NIH) grant no. 1R01CA226537.