p38MAPK builds a hyaluronan cancer niche to drive lung tumorigenesis.

Expansion of neoplastic lesions generates the initial signal that instigates the creation of a tumor niche. Nontransformed cell types within the microenvironment continuously coevolve with tumor cells to promote tumorigenesis. Here, we identify p38MAPK as a key component of human lung cancer, and specifically stromal interactomes, which provides an early, protumorigenic signal in the tissue microenvironment. We found that lung cancer growth depends on short-distance cues produced by the cancer niche in a p38-dependent manner. We identified fibroblast-specific hyaluronan synthesis at the center of p38-driven tumorigenesis, which regulates early stromal fibroblast activation, the conversion to carcinoma-associated fibroblasts (CAFs), and cancer cell proliferation. Systemic down-regulation of p38MAPK signaling in a knock-in model with substitution of activating Tyr182 to phenylalanine or conditional ablation of p38 in fibroblasts has a significant tumor-suppressive effect on K-ras lung tumorigenesis. Furthermore, both Kras-driven mouse lung tumors and orthotopically grown primary human lung cancers show a significant sensitivity to both a chemical p38 inhibitor and an over-the-counter inhibitor of hyaluronan synthesis. We propose that p38MAPK-hyaluronan-dependent reprogramming of the tumor microenvironment plays a critical role in driving lung tumorigenesis, while blocking this process could have far-reaching therapeutic implications.

Long Range Endocrine Delivery of Circulating miR-210 to Endothelium Promotes Pulmonary Hypertension.

RATIONALE: Unproven theories abound regarding the long-range uptake and endocrine activity of extracellular blood-borne microRNAs into tissue. In pulmonary hypertension (PH), microRNA-210 (miR-210) in pulmonary endothelial cells promotes disease, but its activity as an extracellular molecule is incompletely defined. OBJECTIVE: We investigated whether chronic and endogenous endocrine delivery of extracellular miR-210 to pulmonary vascular endothelial cells promotes PH. METHODS AND RESULTS: Using miR-210 replete (wild-type [WT]) and knockout mice, we tracked blood-borne miR-210 using bone marrow transplantation and parabiosis (conjoining of circulatory systems). With bone marrow transplantation, circulating miR-210 was derived predominantly from bone marrow. Via parabiosis during chronic hypoxia to induce miR-210 production and PH, miR-210 was undetectable in knockout-knockout mice pairs. However, in plasma and lung endothelium, but not smooth muscle or adventitia, miR-210 was observed in knockout mice of WT-knockout pairs. This was accompanied by downregulation of miR-210 targets ISCU (iron-sulfur assembly proteins)1/2 and COX10 (cytochrome c oxidase assembly protein-10), indicating endothelial import of functional miR-210. Via hemodynamic and histological indices, knockout-knockout pairs were protected from PH, whereas knockout mice in WT-knockout pairs developed PH. In particular, pulmonary vascular engraftment of miR-210-positive interstitial lung macrophages was observed in knockout mice of WT-knockout pairs. To address whether engrafted miR-210-positive myeloid or lymphoid cells contribute to paracrine miR-210 delivery, we studied miR-210 knockout mice parabiosed with miR-210 WT; Cx3cr1 knockout mice (deficient in myeloid recruitment) or miR-210 WT; Rag1 knockout mice (deficient in lymphocytes). In both pairs, miR-210 knockout mice still displayed miR-210 delivery and PH, thus demonstrating a pathogenic endocrine delivery of extracellular miR-210. CONCLUSIONS: Endogenous blood-borne transport of miR-210 into pulmonary vascular endothelial cells promotes PH, offering fundamental insight into the systemic physiology of microRNA activity. These results also describe a platform for RNA-mediated crosstalk in PH, providing an impetus for developing blood-based miR-210 technologies for diagnosis and therapy in this disease.

Matrix stiffening induces a pathogenic QKI-miR-7-SRSF1 signaling axis in pulmonary arterial endothelial cells.

Pulmonary arterial hypertension (PAH) refers to a set of heterogeneous vascular diseases defined by elevation of pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR), leading to right ventricular (RV) remodeling and often death. Early increases in pulmonary artery stiffness in PAH drive pathogenic alterations of pulmonary arterial endothelial cells (PAECs), leading to vascular remodeling. Dysregulation of microRNAs can drive PAEC dysfunction. However, the role of vascular stiffness in regulating pathogenic microRNAs in PAH is incompletely understood. Here, we demonstrated that extracellular matrix (ECM) stiffening downregulated miR-7 levels in PAECs. The RNA-binding protein quaking (QKI) has been implicated in the biogenesis of miR-7. Correspondingly, we found that ECM stiffness upregulated QKI, and QKI knockdown led to increased miR-7. Downstream of the QKI-miR-7 axis, the serine and arginine-rich splicing factor 1 (SRSF1) was identified as a direct target of miR-7. Correspondingly, SRSF1 was reciprocally upregulated in PAECs exposed to stiff ECM and was negatively correlated with miR-7. Decreased miR-7 and increased QKI and SRSF1 were observed in lungs from patients with PAH and PAH rats exposed to SU5416/hypoxia. Lastly, miR-7 upregulation inhibited human PAEC migration, whereas forced SRSF1 expression reversed this phenotype, proving that miR-7 depended upon SRSF1 to control migration. In aggregate, these results define the QKI-miR-7-SRSF1 axis as a mechanosensitive mechanism linking pulmonary arterial vascular stiffness to pathogenic endothelial function. These findings emphasize implications relevant to PAH and suggest the potential benefit of developing therapies that target this miRNA-dependent axis in PAH.

Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in KCNK3 (potassium channel subfamily K member 3) gene, which encodes an outward rectifier K+ channel, have been identified in pulmonary arterial hypertension patients. OBJECTIVE: We have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature. METHODS AND RESULTS: Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 of Kcnk3 gene and characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, and molecular levels to decipher the cellular mechanisms associated with loss of KCNK3. Using patch-clamp technique, we validated our transgenic strategy by demonstrating the absence of KCNK3 current in freshly isolated pulmonary arterial smooth muscle cells from Kcnk3-mutated rats. At 4 months of age, echocardiographic parameters revealed shortening of the pulmonary artery acceleration time associated with elevation of the right ventricular systolic pressure. Kcnk3-mutated rats developed more severe PH than wild-type rats after monocrotaline exposure or chronic hypoxia exposure. Kcnk3-mutation induced a lung distal neomuscularization and perivascular extracellular matrix activation. Lungs of Kcnk3-mutated rats were characterized by overactivation of ERK1/2 (extracellular signal-regulated kinase1-/2), AKT (protein kinase B), SRC, and overexpression of HIF1-α (hypoxia-inducible factor-1 α), survivin, and VWF (Von Willebrand factor). Linked with plasma membrane depolarization, reduced endothelial-NOS expression and desensitization of endothelial-derived hyperpolarizing factor, Kcnk3-mutated rats presented predisposition to vasoconstriction of pulmonary arteries and a severe loss of sildenafil-induced pulmonary arteries relaxation. Moreover, we showed strong alteration of right ventricular cardiomyocyte excitability. Finally, Kcnk3-mutated rats developed age-dependent PH associated with low serum-albumin concentration. CONCLUSIONS: We established the first Kcnk3-mutated rat model of PH. Our results confirm that KCNK3 loss of function is a key event in pulmonary arterial hypertension pathogenesis. This model presents new opportunities for understanding the initiating mechanisms of PH and testing biologically relevant therapeutic molecules in the context of PH.

UBTD1 is a mechano-regulator controlling cancer aggressiveness.

Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with ß-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase ß-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

Bmpr2 Mutant Rats Develop Pulmonary and Cardiac Characteristics of Pulmonary Arterial Hypertension.

BACKGROUND: Monoallelic mutations in the gene encoding bone morphogenetic protein receptor 2 ( Bmpr2) are the main genetic risk factor for heritable pulmonary arterial hypertension (PAH) with incomplete penetrance. Several Bmpr2 transgenic mice have been reported to develop mild spontaneous PAH. In this study, we examined whether rats with the Bmpr2 mutation were susceptible to developing more severe PAH. METHODS: The zinc finger nuclease method was used to establish rat lines with mutations in the Bmpr2 gene. These rats were then characterized at the hemodynamic, histological, electrophysiological, and molecular levels. RESULTS: Rats with a monoallelic deletion of 71 bp in exon 1 (Δ 71 rats) showed decreased BMPRII expression and phosphorylated SMAD1/5/9 levels. Δ 71 Rats develop age-dependent spontaneous PAH with a low penetrance (16%-27%), similar to that in humans. Δ 71 Rats were more susceptible to hypoxia-induced pulmonary hypertension than wild-type rats. Δ 71 Rats exhibited progressive pulmonary vascular remodeling associated with a proproliferative phenotype and showed lower pulmonary microvascular density than wild-type rats. Organ bath studies revealed severe alteration of pulmonary artery contraction and relaxation associated with potassium channel subfamily K member 3 (KCNK3) dysfunction. High levels of perivascular fibrillar collagen and pulmonary interleukin-6 overexpression discriminated rats that developed spontaneous PAH and rats that did not develop spontaneous PAH. Finally, detailed assessments of cardiomyocytes demonstrated alterations in morphology, calcium (Ca2+), and cell contractility specific to the right ventricle; these changes could explain the lower cardiac output of Δ 71 rats. Indeed, adult right ventricular cardiomyocytes from Δ 71 rats exhibited a smaller diameter, decreased sensitivity of sarcomeres to Ca2+, decreased [Ca2+] transient amplitude, reduced sarcoplasmic reticulum Ca2+ content, and short action potential duration compared with right ventricular cardiomyocytes from wild-type rats. CONCLUSIONS: We characterized the first Bmpr2 mutant rats and showed some of the critical cellular and molecular dysfunctions described in human PAH. We also identified the heart as an unexpected but potential target organ of Bmpr2 mutations. Thus, this new genetic rat model represents a promising tool to study the pathogenesis of PAH.

BOLA (BolA Family Member 3) Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension.

BACKGROUND: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. METHODS: In vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. RESULTS: In cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2α-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. CONCLUSIONS: BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development.

Inhibition of CHK 1 (Checkpoint Kinase 1) Elicits Therapeutic Effects in Pulmonary Arterial Hypertension.

OBJECTIVE: Pulmonary arterial hypertension (PAH) is a debilitating disease associated with progressive vascular remodeling of distal pulmonary arteries leading to elevation of pulmonary artery pressure, right ventricular hypertrophy, and death. Although presenting high levels of DNA damage that normally jeopardize their viability, pulmonary artery smooth muscle cells (PASMCs) from patients with PAH exhibit a cancer-like proproliferative and apoptosis-resistant phenotype accounting for vascular lumen obliteration. In cancer cells, overexpression of the serine/threonine-protein kinase CHK1 (checkpoint kinase 1) is exploited to counteract the excess of DNA damage insults they are exposed to. This study aimed to determine whether PAH-PASMCs have developed an orchestrated response mediated by CHK1 to overcome DNA damage, allowing cell survival and proliferation. Approach and Results: We demonstrated that CHK1 expression is markedly increased in isolated PASMCs and distal PAs from patients with PAH compared with controls, as well as in multiple complementary animal models recapitulating the disease, including monocrotaline rats and the simian immunodeficiency virus-infected macaques. Using a pharmacological and molecular loss of function approach, we showed that CHK1 promotes PAH-PASMCs proliferation and resistance to apoptosis. In addition, we found that inhibition of CHK1 induces downregulation of the DNA repair protein RAD 51 and severe DNA damage. In vivo, we provided evidence that pharmacological inhibition of CHK1 significantly reduces vascular remodeling and improves hemodynamic parameters in 2 experimental rat models of PAH. CONCLUSIONS: Our results show that CHK1 exerts a proproliferative function in PAH-PASMCs by mitigating DNA damage and suggest that CHK1 inhibition may, therefore, represent an attractive therapeutic option for patients with PAH.

Factors Associated with Heritable Pulmonary Arterial Hypertension Exert Convergent Actions on the miR-130/301-Vascular Matrix Feedback Loop.

Pulmonary arterial hypertension (PAH) is characterized by occlusion of lung arterioles, leading to marked increases in pulmonary vascular resistance. Although heritable forms of PAH are known to be driven by genetic mutations that share some commonality of function, the extent to which these effectors converge to regulate shared processes in this disease is unknown. We have causally connected extracellular matrix (ECM) remodeling and mechanotransduction to the miR-130/301 family in a feedback loop that drives vascular activation and downstream PAH. However, the molecular interconnections between factors genetically associated with PAH and this mechano-driven feedback loop remain undefined. We performed systematic manipulation of matrix stiffness, the miR-130/301 family, and factors genetically associated with PAH in primary human pulmonary arterial cells and assessed downstream and reciprocal consequences on their expression. We found that a network of factors linked to heritable PAH converges upon the matrix stiffening-miR-130/301-PPARγ-LRP8 axis in order to remodel the ECM. Furthermore, we leveraged a computational network biology approach to predict a number of additional molecular circuits functionally linking this axis to the ECM. These results demonstrate that multiple genes associated with heritable PAH converge to control the miR-130/301 circuit, triggering a self-amplifying feedback process central to pulmonary vascular stiffening and disease.

The microRNA-130/301 family controls vasoconstriction in pulmonary hypertension.

Pulmonary hypertension (PH) is a complex disorder, spanning several known vascular cell types. Recently, we identified the microRNA-130/301 (miR-130/301) family as a regulator of multiple pro-proliferative pathways in PH, but the true breadth of influence of the miR-130/301 family across cell types in PH may be even more extensive. Here, we employed targeted network theory to identify additional pathogenic pathways regulated by miR-130/301, including those involving vasomotor tone. Guided by these predictions, we demonstrated, via gain- and loss-of-function experimentation in vitro and in vivo, that miR-130/301-specific control of the peroxisome proliferator-activated receptor γ regulates a panel of vasoactive factors communicating between diseased pulmonary vascular endothelial and smooth muscle cells. Of these, the vasoconstrictive factor endothelin-1 serves as an integral point of communication between the miR-130/301-peroxisome proliferator-activated receptor γ axis in endothelial cells and contractile function in smooth muscle cells. Thus, resulting from an in silico analysis of the architecture of the PH disease gene network coupled with molecular experimentation in vivo, these findings clarify the expanded role of the miR-130/301 family in the global regulation of PH. They further emphasize the importance of molecular cross-talk among the diverse cellular populations involved in PH.

miR-193b/365a cluster controls progression of epidermal squamous cell carcinoma.

Incidence of cutaneous squamous cell carcinomas (cSCCs) constantly increases in the Caucasian population. Developing preferentially on precancerous lesions such as actinic keratoses due to chronic sunlight exposure, cSCCs result from the malignant transformation of keratinocytes. Although a resection of the primary tumor is usually curative, a subset of aggressive cSCCs shows a high risk of recurrence and metastases. The characterization of the molecular dysfunctions involved in cSCC development should help to identify new relevant targets against these aggressive cSCCs. In that context, we have used small RNA sequencing to identify 100 microRNAs (miRNAs) whose expression was altered during chemically induced mouse skin tumorigenesis. The decreased expression of the miR-193b/365a cluster during tumor progression suggests a tumor suppressor role. Ectopic expression of these miRNAs in tumor cells indeed inhibited their proliferation, clonogenic potential and migration, which were stimulated in normal keratinocytes when these miRNAs were blocked with antisense oligonucleotides. A combination of in silico predictions and transcriptome analyses identified several target genes of interest. We validated KRAS and MAX as direct targets of miR-193b and miR-365a. Repression of these targets using siRNAs mimicked the effects of miR-193b and miR-365a, suggesting that these genes might mediate, at least in part, the tumor-suppressive action of these miRNAs.

Pulmonary primary oxysterol and bile acid synthesis as a predictor of outcomes in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare and fatal vascular disease with heterogeneous clinical manifestations. To date, molecular determinants underlying the development of PAH and related outcomes remain poorly understood. Herein, we identify pulmonary primary oxysterol and bile acid synthesis (PPOBAS) as a previously unrecognized pathway central to PAH pathophysiology. Mass spectrometry analysis of 2,756 individuals across five independent studies revealed 51 distinct circulating metabolites that predicted PAH-related mortality and were enriched within the PPOBAS pathway. Across independent single-center PAH studies, PPOBAS pathway metabolites were also associated with multiple cardiopulmonary measures of PAH-specific pathophysiology. Furthermore, PPOBAS metabolites were found to be increased in human and rodent PAH lung tissue and specifically produced by pulmonary endothelial cells, consistent with pulmonary origin. Finally, a poly-metabolite risk score comprising 13 PPOBAS molecules was found to not only predict PAH-related mortality but also outperform current clinical risk scores. This work identifies PPOBAS as specifically altered within PAH and establishes needed prognostic biomarkers for guiding therapy in PAH.

Dietary intake and glutamine-serine metabolism control pathologic vascular stiffness.

Perivascular collagen deposition by activated fibroblasts promotes vascular stiffening and drives cardiovascular diseases such as pulmonary hypertension (PH). Whether and how vascular fibroblasts rewire their metabolism to sustain collagen biosynthesis remains unknown. Here, we found that inflammation, hypoxia, and mechanical stress converge on activating the transcriptional coactivators YAP and TAZ (WWTR1) in pulmonary arterial adventitial fibroblasts (PAAFs). Consequently, YAP and TAZ drive glutamine and serine catabolism to sustain proline and glycine anabolism and promote collagen biosynthesis. Pharmacologic or dietary intervention on proline and glycine anabolic demand decreases vascular stiffening and improves cardiovascular function in PH rodent models. By identifying the limiting metabolic pathways for vascular collagen biosynthesis, our findings provide guidance for incorporating metabolic and dietary interventions for treating cardiopulmonary vascular disease.

Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension.

Vascular inflammation critically regulates endothelial cell (EC) pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulation of lysosomal activity and cholesterol metabolism have known inflammatory roles in disease, but their relevance to PAH is unclear. In human pulmonary arterial ECs and in PAH, we found that inflammatory cytokine induction of the nuclear receptor coactivator 7 (NCOA7) both preserved lysosomal acidification and served as a homeostatic brake to constrain EC immunoactivation. Conversely, NCOA7 deficiency promoted lysosomal dysfunction and proinflammatory oxysterol/bile acid generation that, in turn, contributed to EC pathophenotypes. In vivo, mice deficient for Ncoa7 or exposed to the inflammatory bile acid 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) displayed worsened PAH. Emphasizing this mechanism in human PAH, an unbiased, metabolome-wide association study (N=2,756) identified a plasma signature of the same NCOA7-dependent oxysterols/bile acids associated with PAH mortality (P<1.1x10-6). Supporting a genetic predisposition to NCOA7 deficiency, in genome-edited, stem cell-derived ECs, the common variant intronic SNP rs11154337 in NCOA7 regulated NCOA7 expression, lysosomal activity, oxysterol/bile acid production, and EC immunoactivation. Correspondingly, SNP rs11154337 was associated with PAH severity via six-minute walk distance and mortality in discovery (N=93, P=0.0250; HR=0.44, 95% CI [0.21-0.90]) and validation (N=630, P=2x10-4; HR=0.49, 95% CI [0.34-0.71]) cohorts. Finally, utilizing computational modeling of small molecule binding to NCOA7, we predicted and synthesized a novel activator of NCOA7 that prevented EC immunoactivation and reversed indices of rodent PAH. In summary, we have established a genetic and metabolic paradigm and a novel therapeutic agent that links lysosomal biology as well as oxysterol and bile acid processes to EC inflammation and PAH pathobiology. This paradigm carries broad implications for diagnostic and therapeutic development in PAH and in other conditions dependent upon acquired and innate immune regulation of vascular disease.

Frataxin deficiency disrupts mitochondrial respiration and pulmonary endothelial cell function.

Deficiency of iron­sulfur (FeS) clusters promotes metabolic rewiring of the endothelium and the development of pulmonary hypertension (PH) in vivo. Joining a growing number of FeS biogenesis proteins critical to pulmonary endothelial function, recent data highlighted that frataxin (FXN) reduction drives Fe-S-dependent genotoxic stress and senescence across multiple types of pulmonary vascular disease. Trinucleotide repeat mutations in the FXN gene cause Friedreich's ataxia, a disease characterized by cardiomyopathy and neurodegeneration. These tissue-specific phenotypes have historically been attributed to mitochondrial reprogramming and oxidative stress. Whether FXN coordinates both nuclear and mitochondrial processes in the endothelium is unknown. Here, we aim to identify the mitochondria-specific effects of FXN deficiency in the endothelium that predispose to pulmonary hypertension. Our data highlight an Fe-S-driven metabolic shift separate from previously described replication stress whereby FXN knockdown diminished mitochondrial respiration and increased glycolysis and oxidative species production. In turn, FXN-deficient endothelial cells had increased vasoconstrictor production (EDN1) and decreased nitric oxide synthase expression (NOS3). These data were observed in primary pulmonary endothelial cells after pharmacologic inhibition of FXN, mice carrying a genetic endothelial deletion of FXN, and inducible pluripotent stem cell-derived endothelial cells from patients with FXN mutations. Altogether, this study indicates FXN is an upstream driver of pathologic aberrations in metabolism and genomic stability. Moreover, our study highlights FXN-specific vasoconstriction in vivo, prompting future studies to investigate available and novel PH therapies in contexts of FXN deficiency.

Mechano-dependent sorbitol accumulation supports biomolecular condensate.

Biomolecular condensates regulate a wide range of cellular functions from signaling to RNA metabolism 1, 2 , yet, the physiologic conditions regulating their formation remain largely unexplored. Biomolecular condensate assembly is tightly regulated by the intracellular environment. Changes in the chemical or physical conditions inside cells can stimulate or inhibit condensate formation 3-5 . However, whether and how the external environment of cells can also regulate biomolecular condensation remain poorly understood. Increasing our understanding of these mechanisms is paramount as failure to control condensate formation and dynamics can lead to many diseases 6, 7 . Here, we provide evidence that matrix stiffening promotes biomolecular condensation in vivo . We demonstrate that the extracellular matrix links mechanical cues with the control of glucose metabolism to sorbitol. In turn, sorbitol acts as a natural crowding agent to promote biomolecular condensation. Using in silico simulations and in vitro assays, we establish that variations in the physiological range of sorbitol, but not glucose, concentrations, are sufficient to regulate biomolecular condensates. Accordingly, pharmacologic and genetic manipulation of intracellular sorbitol concentration modulates biomolecular condensates in breast cancer - a mechano-dependent disease. We propose that sorbitol is a mechanosensitive metabolite enabling protein condensation to control mechano-regulated cellular functions. Altogether, we uncover molecular driving forces underlying protein phase transition and provide critical insights to understand the biological function and dysfunction of protein phase separation.

Single Nucleotide Polymorphism rs9277336 Controls the Nuclear Alpha Actinin 4-Human Leukocyte Antigen-DPA1 Axis and Pulmonary Endothelial Pathophenotypes in Pulmonary Arterial Hypertension.

Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the All of Us database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium (r2>0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele-specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA-DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA-DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4 and controlling expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.

miR-483-3p controls proliferation in wounded epithelial cells.

The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in 2 distinct models of wound healing: scratch-injured cultures of human keratinocytes and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-2'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits keratinocyte migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of keratinocytes transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR-483-3p that control keratinocyte proliferation. miR-483-3p-mediated down-regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.

Metabo-reciprocity in cell mechanics: feeling the demands/feeding the demand.

Altered metabolic programs and corruption of tissue architecture are hallmarks of disease. The spatiotemporal control of cell behavior requires transmission of information from the complex structure of tissues to their constituent cells. Cytoskeletal mechanotransduction enables this transmission by sensing mechanical environments and adapting cellular behaviors. However, this process requires energy. Recent findings have shed light on the bidirectional relationship between mechanical forces and upstream and downstream metabolic cues. We discuss recent advances in the reciprocal regulation ('metabo-reciprocity') that allows cells to adapt their metabolic needs to their mechanically constrained environment but can also contribute to adjustable feedback that promotes disease progression.

Simultaneous Pharmacologic Inhibition of Yes-Associated Protein 1 and Glutaminase 1 via Inhaled Poly(Lactic-co-Glycolic) Acid-Encapsulated Microparticles Improves Pulmonary Hypertension.

Background Pulmonary hypertension (PH) is a deadly disease characterized by vascular stiffness and altered cellular metabolism. Current treatments focus on vasodilation and not other root causes of pathogenesis. Previously, it was demonstrated that glutamine metabolism, as catalyzed by GLS1 (glutaminase 1) activity, is mechanoactivated by matrix stiffening and the transcriptional coactivators YAP1 (yes-associated protein 1) and transcriptional coactivator with PDZ-binding motif (TAZ), resulting in pulmonary vascular proliferation and PH. Pharmacologic inhibition of YAP1 (by verteporfin) or glutaminase (by CB-839) improved PH in vivo. However, systemic delivery of these agents, particularly YAP1 inhibitors, may have adverse chronic effects. Furthermore, simultaneous use of pharmacologic blockers may offer additive or synergistic benefits. Therefore, a strategy that delivers these drugs in combination to local lung tissue, thus avoiding systemic toxicity and driving more robust improvement, was investigated. Methods and Results We used poly(lactic-co-glycolic) acid polymer-based microparticles for delivery of verteporfin and CB-839 simultaneously to the lungs of rats suffering from monocrotaline-induced PH. Microparticles released these drugs in a sustained fashion and delivered their payload in the lungs for 7 days. When given orotracheally to the rats weekly for 3 weeks, microparticles carrying this drug combination improved hemodynamic (right ventricular systolic pressure and right ventricle/left ventricle+septum mass ratio), histologic (vascular remodeling), and molecular markers (vascular proliferation and stiffening) of PH. Importantly, only the combination of drug delivery, but neither verteporfin nor CB-839 alone, displayed significant improvement across all indexes of PH. Conclusions Simultaneous, lung-specific, and controlled release of drugs targeting YAP1 and GLS1 improved PH in rats, addressing unmet needs for the treatment of this deadly disease.