Pathophysiology of hypoxia: molecular mechanisms involved in pulmonary hypertension and renal carcinoma

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 21-04-2017Hypoxia is a well-studied prototype and a paradigm of responses that involve the organism as a whole with different consequences on health and disease. Prolonged generalized hypoxia is the cause of hypoxic pulmonary arterial hypertension (PAH). Conversely hypoxia can also be a consequence during uncontrolled growth of tumors with aberrant blood vessels and thus an insufficient oxygen supply. In this work, hypoxia was treated from two points of view, as the cause in hypoxic PAH or as the consequence of cancer in clear cell renal cell carcinoma (ccRCC). Hypoxic conditions stimulate pulmonary vasoconstriction and vascular remodeling, both pathognomonic changes in PAH. The secreted protein thrombospondin-1 (TSP1) promotes PAH, but it is largely unknown how hypoxia regulates TSP1 in the lung and whether this contributes to pathological events in PAH. Using a murine model of constitutive hypoxia, gene silencing and luciferase reporter experiments in cells, we found that hypoxia induced pulmonary TSP1 in a HIF2α-dependent manner. Additionally, we found that TSP1 induced migration in pulmonary fibroblasts and PASMC, and increased permeability in PAEC through effects on the cell-matrix and cell-cell interactions. In fibroblasts, TSP1 induced differentiation into a SMC-like phenotype, while in PASMC, TSP1 seems to have a role in the regulation of the potassium channel Kv1.5. In addition, we found in end-stage PAH patients, both TSP1 and HIF2α protein expression increased in pulmonary arteries compared to non-PAH controls. This provides genetic evidence that HIF2α-induced TSP1 contributes to vascular response and remodeling during PAH. 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 studies in ccRCC demonstrated that loss of VHL, hypoxia or PHD inactivation decreased VCAM-1 levels through a transcriptional mechanism that was independent of the hypoxia-induced transcription factor HIF and dependent on the NF-κB signaling pathway. VCAM-1 decreased levels in ccRCC might be relevant to tumor cell immune evasion since this molecule elicited immune cell binding specifically mediated by its interaction with the α4β1 integrin expressed in the immune cells. Furthermore, this interaction made ccRCC cells more sensitive to the cytotoxic effects mediated by activated monocytic cells. Remarkably, in ccRCC human samples with VHL non-missense mutations we observed a negative correlation between VCAM-1 levels and ccRCC stage and microvascular invasion pointing out the clinical value of VCAM-1 levels as a marker of ccRCC progression.La hipoxia es un proceso bien estudiado y un paradigma de respuestas que involucran al organismo como un todo con diferentes consecuencias sobre la salud y la enfermedad. La hipoxia generalizada prolongada es la causa de la hipertensión arterial pulmonar (HAP) inducida por hipoxia. Por el contrario, la hipoxia también puede ser una consecuencia durante el crecimiento descontrolado de tumores con vasos sanguíneos aberrantes y, por tanto, un suministro insuficiente de oxígeno. En este trabajo, la hipoxia se ha tratado desde dos puntos de vista, como causa en HAP hipóxica o como consecuencia en el carcinoma renal de célula clara (ccRCC). La hipoxia estimula la vasoconstricción pulmonar y el remodelado vascular, cambios patognomónicos en la HAP. La proteína secretada trombospondina-1 (TSP1) promueve la HAP, pero se desconoce cómo la hipoxia regula su expresión en pulmón y si esto contribuye a los eventos patológicos en la HAP. Usando un modelo murino de hipoxia constitutiva, silenciamiento génico y experimentos reporteros, encontramos que la hipoxia inducía TSP1 en pulmón de manera dependiente de HIF2α. Además, se encontró que TSP1 inducía la migración de fibroblastos pulmonares y PASMC, y aumentaba la permeabilidad en PAEC, a través de efectos sobre las interacciones célula-matriz y célula-célula. En los fibroblastos, TSP1 indujo la diferenciación a un fenotipo de tipo SMC y en PASMC TSP1 parece tener un papel en la regulación del canal de potasio Kv1.5. Además, en pacientes en estado terminal de HAP se encontraron altos niveles de TSP1 y HIF2α comparados con controles sanos. Estos resultados proporcionan evidencias genéticas de que la TSP1 inducida por hipoxia contribuye a la respuesta vascular y al remodelado durante la HAP. VCAM-1 es una molécula de adhesión asignada al endotelio activado que media la adhesión y la extravasación de las células inmunes. Sin embargo, su expresión en carcinoma renal se correlaciona inversamente con tumores malignos. Nuestros estudios en ccRCC demostraron que la pérdida de VHL, la hipoxia o la inactivación de las PHDs disminuían los niveles de VCAM-1 a través de un mecanismo de transcripción que era independiente del factor de transcripción HIF y dependiente de la vía de señalización NF-κB. Los niveles disminuidos de VCAM-1 en ccRCC podrían ser relevantes para la evasión inmune de células tumorales, ya que la interacción de esta molécula con su receptor, la integrina α4β1 expresada en células inmunes, provocó la unión de las células inmunes con las células ccRCC. Además, esta interacción hizo que las células ccRCC fueran más sensibles a los efectos citotóxicos mediados por células monocíticas activadas. Es destacable que en muestras humanas de ccRCC con mutaciones sin sentido en vhl se observó una correlación negativa entre los niveles de VCAM-1 y el estadio de ccRCC y la invasión microvascular señalando el valor clínico de los niveles de VCAM-1 como marcador de progresión de ccRCC

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.This work was supported by grants from the Spanish Government (co-funded by European Regional Development Fund, ERDF/FEDER); PI16/02166 and 2017/EEUU/03 to MJC; Red Temática de Excelencia en Investigación en Hipoxia (SAF 2017-90794-REDT) to MJ

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

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 α4β1 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.This work was supported by grants from the Instituto de Salud Carlos III (co-funded by the European Union and Fondo Europeo de Desarrollo Regional; grants PI16/02166 and PIE13/00041), a grant from Red Cardiovascular (RD12/0042/0065 to M.J. Calzada), and a grant from Ministerio de Economía y competitividad (SAF2015-64215R to R. Sánchez-Prieto).Peer reviewe