Cigarette Smoke Directly Promotes Pulmonary Arterial Remodeling and Kv7.4 Channel Dysfunction.

Rationale: Cigarette smoke is considered the chief leading cause of chronic obstructive pulmonary disease (COPD). Its impact on the progressive deterioration of airways has been extensively studied, but its direct effects on the pulmonary vasculature are less known. Objectives: To prove that pulmonary arterial remodeling in patients with COPD is not just a consequence of alveolar hypoxia but also due to the direct effects of cigarette smoke on the pulmonary vascular bed. Methods: We have used different molecular and cell biology approaches, as well as traction force microscopy, wire myography, and patch-clamp techniques in human cells and freshly isolated pulmonary arteries. In addition, we relied on in vivo models and human samples to analyze the effects of cigarette smoke on pulmonary vascular tone alterations. Measurements and Main Results: Cigarette smoke extract exposure directly promoted a hypertrophic, senescent phenotype that in turn contributed, through the secretion of inflammatory molecules, to an increase in the proliferative potential of nonexposed cells. Interestingly, these effects were significantly reversed by antioxidants. Furthermore, cigarette smoke extract affected cell contractility and dysregulated the expression and activity of the voltage-gated K+ channel Kv7.4. This contributed to the impairment of vasoconstriction and vasodilation responses. Most importantly, the levels of this channel were diminished in the lungs of smoke-exposed mice, smokers, and patients with COPD. Conclusions: Cigarette smoke directly contributes to pulmonary arterial remodeling through increased cell senescence, as well as vascular tone alterations because of diminished levels and function in the Kv7.4 channel. Strategies targeting these pathways may lead to novel therapies for COPD.

Friction forces determine cytoplasmic reorganization and shape changes of ascidian oocytes upon fertilization.

Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole-a protuberance of the zygote's vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces.

pVHL-mediated regulation of the anti-angiogenic protein thrombospondin-1 decreases migration of Clear Cell Renal Carcinoma Cell Lines.

Thrombospondin-1 (TSP-1) is a multifunctional matrix protein with antitumor activities due in part to its ability to inhibit angiogenesis, which in turn contributes to determine the fate of many tumours. Previous studies have shown that TSP-1 expression supports normal kidney angiostasis, and decreased TSP-1 levels contribute to the angiogenic phenotype of renal cell carcinomas (RCC). The loss of the von Hippel-Lindau tumour suppressor gene (VHL) in these tumours favours stabilization of the Hypoxia Inducible Factors (HIF), which in turn contribute to adapt tumour cells to hostile environments promoting tumour progression. However, HIF-independent regulation of certain genes might also be involved. We have previously shown that TSP-1 is regulated in hypoxia in clear cell RCC (ccRCC) in a HIF-independent manner; however, the effect of VHL protein (pVHL) on TSP-1 expression has not been evaluated. Our results proved that pVHL loss or mutation in its alpha or beta domain significantly decreased TSP-1 levels in ccRCC in a HIF-independent manner. Furthermore, this regulation proved to be important for ccRCC cells behaviour showing that decreased TSP-1 levels rendered ccRCC cells more migratory. This data substantiates a unique regulation pattern for TSP-1 in a pVHL-dependent manner, which may be relevant in the aggressiveness of ccRCC.

Hypoxia and Redox Signaling on Extracellular Matrix Remodeling: From Mechanisms to Pathological Implications.

SIGNIFICANCE: The extracellular matrix (ECM) is an essential modulator of cell behavior that influences tissue organization. It has a strong relevance in homeostasis and translational implications for human disease. In addition to ECM structural proteins, matricellular proteins are important regulators of the ECM that are involved in a myriad of different pathologies. Recent Advances: Biochemical studies, animal models, and study of human diseases have contributed to the knowledge of molecular mechanisms involved in remodeling of the ECM, both in homeostasis and disease. Some of them might help in the development of new therapeutic strategies. This review aims to review what is known about some of the most studied matricellular proteins and their regulation by hypoxia and redox signaling, as well as the pathological implications of such regulation. CRITICAL ISSUES: Matricellular proteins have complex regulatory functions and are modulated by hypoxia and redox signaling through diverse mechanisms, in some cases with controversial effects that can be cell or tissue specific and context dependent. Therefore, a better understanding of these regulatory processes would be of great benefit and will open new avenues of considerable therapeutic potential. FUTURE DIRECTIONS: Characterizing the specific molecular mechanisms that modulate matricellular proteins in pathological processes that involve hypoxia and redox signaling warrants additional consideration to harness the potential therapeutic value of these regulatory proteins. Antioxid. Redox Signal. 27, 802-822.

VHL promotes immune response against renal cell carcinoma via NF-κB-dependent regulation of VCAM-1.

Vascular cell adhesion molecule 1 (VCAM-1) is an adhesion molecule assigned to the activated endothelium mediating immune cells adhesion and extravasation. However, its expression in renal carcinomas inversely correlates with tumor malignancy. Our experiments in clear cell renal cell carcinoma (ccRCC) cell lines demonstrated that von Hippel Lindau (VHL) loss, hypoxia, or PHD (for prolyl hydroxylase domain-containing proteins) inactivation decreased VCAM-1 levels through a transcriptional mechanism that was independent of the hypoxia-inducible factor and dependent on the nuclear factor κB signaling pathway. Conversely, VHL expression leads to high VCAM-1 levels in ccRCC, which in turn leads to better outcomes, possibly by favoring antitumor immunity through VCAM-1 interaction with the α4ß1 integrin expressed in immune cells. Remarkably, in ccRCC human samples with VHL nonmissense mutations, we observed a negative correlation between VCAM-1 levels and ccRCC stage, microvascular invasion, and symptom presentation, pointing out the clinical value of VCAM-1 levels as a marker of ccRCC progression.

TSP1-CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction.

AIMS: Thrombospondin-1 (TSP1) is a ligand for CD47 and TSP1-/- mice are protected from pulmonary hypertension (PH). We hypothesized the TSP1-CD47 axis is upregulated in human PH and promotes pulmonary arterial vasculopathy. METHODS AND RESULTS: We analyzed the molecular signature and functional response of lung tissue and distal pulmonary arteries (PAs) from individuals with (n = 23) and without (n = 16) PH. Compared with controls, lungs and distal PAs from PH patients showed induction of TSP1-CD47 and endothelin-1/endothelin A receptor (ET-1/ETA) protein and mRNA. In control PAs, treatment with exogenous TSP1 inhibited vasodilation and potentiated vasoconstriction to ET-1. Treatment of diseased PAs from PH patients with a CD47 blocking antibody improved sensitivity to vasodilators. Hypoxic wild type (WT) mice developed PH and displayed upregulation of pulmonary TSP1, CD47, and ET-1/ETA concurrent with down regulation of the transcription factor cell homolog of the v-myc oncogene (cMyc). In contrast, PH was attenuated in hypoxic CD47-/- mice while pulmonary TSP1 and ET-1/ETA were unchanged and cMyc was overexpressed. In CD47-/- pulmonary endothelial cells cMyc was increased and ET-1 decreased. In CD47+/+ cells, forced induction of cMyc suppressed ET-1 transcript, whereas suppression of cMyc increased ET-1 signaling. Furthermore, disrupting TSP1-CD47 signaling in pulmonary smooth muscle cells abrogated ET-1-stimulated hypertrophy. Finally, a CD47 antibody given 2 weeks after monocrotaline challenge in rats upregulated pulmonary cMyc and improved aberrations in PH-associated cardiopulmonary parameters. CONCLUSIONS: In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1-CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

HIF-2α-mediated induction of pulmonary thrombospondin-1 contributes to hypoxia-driven vascular remodelling and vasoconstriction.

AIMS: Hypoxic conditions stimulate pulmonary vasoconstriction and vascular remodelling, both pathognomonic changes in pulmonary arterial hypertension (PAH). The secreted protein thrombospondin-1 (TSP1) is involved in the maintenance of lung homeostasis. New work identified a role for TSP1 in promoting PAH. Nonetheless, it is largely unknown how hypoxia regulates TSP1 in the lung and whether this contributes to pathological events during PAH. METHODS AND RESULTS: In cell and animal experiments, we found that hypoxia induces TSP1 in lungs, pulmonary artery smooth muscle cells and endothelial cells, and pulmonary fibroblasts. Using a murine model of constitutive hypoxia, gene silencing, and luciferase reporter experiments, we found that hypoxia-mediated induction of pulmonary TSP1 is a hypoxia-inducible factor (HIF)-2α-dependent process. Additionally, hypoxic tsp1(-/-) pulmonary fibroblasts and pulmonary artery smooth muscle cell displayed decreased migration compared with wild-type (WT) cells. Furthermore, hypoxia-mediated induction of TSP1 destabilized endothelial cell-cell interactions. This provides genetic evidence that TSP1 contributes to vascular remodelling during PAH. Expanding cell data to whole tissues, we found that, under hypoxia, pulmonary arteries (PAs) from WT mice had significantly decreased sensitivity to acetylcholine (Ach)-stimulated endothelial-dependent vasodilation. In contrast, hypoxic tsp1(-/-) PAs retained sensitivity to Ach, mediated in part by TSP1 regulation of pulmonary Kv channels. Translating these preclinical studies, we find in the lungs from individuals with end-stage PAH, both TSP1 and HIF-2α protein expression increased in the pulmonary vasculature compared with non-PAH controls. CONCLUSIONS: These findings demonstrate that HIF-2α is clearly implicated in the TSP1 pulmonary regulation and provide new insights on its contribution to PAH-driven vascular remodelling and vasoconstriction.