Neurotrophic Factor Mediated Neurorestorative Effect of Pongamia pinnata Against Transient Cerebral Hypoperfusion and Reperfusion in Rats: Preliminary Histological and Molecular Level Evidence / Efecto Neurorestaurador Mediado por Factor Neurotrófico de Pongamia pinnata Contra la Hipoperfusión y Reperfusión Cerebral Transitoria en Ratas: Evidencia Preliminar de un Estudio a Nivel Histológico y Molecular

SUMMARY: Stroke is the leading cause of acquired physical disability in adults and second leading cause of mortality throughout the world. Treatment strategies to curb the effects of stroke would be of great benefit. Pongamia pinnata is a recent attraction in medicine, owing to its abundant medicinal benefits with minimal side effects. The present study aimed to examine acute and subacute effect of Pongamia pinnata leaf extract on transient cerebral hypoperfusion and reperfusion (tCHR) in Wistar rats. 24 adult Wistar rats (12 each for acute and subacute study) were divided in to four groups each viz normal control group, tCHR + NS group, tCHR + 200mg/kg bw and tCHR + 400mg/kg bw groups. Cerebral ischemia induction was carried out by bilateral common carotid artery occlusion and reperfusion. Ethanolic extract of Pongamia pinnata leaves were orally administered for 7 days and 21 days after the surgical procedure for acute and subacute study respectively. Behavioural analysis, histological assessment, and estimation of mRNA levels of HIF-1, GDNF, BDNF and NF-kB were performed. In both acute and subacute study, there was significant improvement in the beam walking assay, neuronal count, decreased neuronal damage in histological sections and higher mRNA expression of BDNF and GDNF in the treatment groups. There was no significant difference in the expression of HIF1 and NF-kB. Thus, Pongamia pinnata has excellent neurorestorative property reversing many of the effects of ischemic stroke induced by tCHR in rats with the underlying mechanism being an improvement in the expression of neurotrophic factors GDNF and BDNF.

El ataque cerebrovascular es la principal causa de discapacidad física adquirida en adultos y la segunda causa de mortalidad en todo el mundo. Las estrategias de tratamiento para frenar los efectos del ataque cerebrovascular serían de gran beneficio. Pongamia pinnata es una atracción reciente en la medicina, debido a sus abundantes beneficios medicinales con mínimos efectos secundarios. El presente estudio tuvo como objetivo examinar el efecto agudo y subagudo del extracto de hoja de Pongamia pinnata sobre la hipoperfusión y reperfusión cerebral transitoria (tCHR) en ratas Wistar. Se dividieron 24 ratas Wistar adultas (12 cada una para el estudio agudo y subagudo) en cuatro grupos, el grupo control normal, el grupo tCHR + NS, los grupos tCHR + 200 mg/kg de peso corporal y tCHR + 400 mg/kg de peso corporal. La inducción de la isquemia cerebral se llevó a cabo mediante oclusión y reperfusión bilateral de la arteria carótida común. El extracto etanólico de hojas de Pongamia pinnata se administró por vía oral durante 7 días y 21 días después del procedimiento quirúrgico para estudio agudo y subagudo respectivamente. Se realizaron análisis de comportamiento, evaluación histológica y estimación de los niveles de ARNm de HIF-1, GDNF, BDNF y NF-kB. Tanto en el estudio agudo como en el subagudo, hubo una mejora significativa en el ensayo de desplazamiento del haz, el recuento neuronal, una disminución del daño neuronal en las secciones histológicas y una mayor expresión de ARNm de BDNF y GDNF en los grupos con tratamiento. No hubo diferencias significativas en la expresión de HIF1 y NF-kB. Por lo tanto, Pongamia pinnata tiene una excelente propiedad neurorestauradora que revierte muchos de los efectos del ataque cerebrovascular isquémico inducido por tCHR en ratas, siendo el mecanismo subyacente una mejora en la expresión de los factores neurotróficos GDNF y BDNF.

Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS: We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS: Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and ß-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and ß-catenin and protein level of ß-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of ß-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION: bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

Mecanismo sensor y de adaptación a los niveles de oxígeno y su implicancia en las enfermedades cardiovasculares: a propósito del Premio Nobel de Fisiología-Medicina 2019 / Role of hypoxia-inducible factor-1 (HIF-1) in the adaptacion to oxygen levels: Nobel Prize in physiology-medicine 2019

RESUMEN: El Premio Nobel 2019 en Fisiología-Medicina se confirió a los Profesores Gregg Semenza, William Kaelin y Sir Peter Ratcliffe por sus investigaciones en la maquinaria molecular que regula la expresión de genes sensibles a los cambios en los niveles de oxígeno. La síntesis de eritropoyetina inducida por la disminución de los niveles sanguíneos de oxígeno condujo al estudio del gen de la eritropoyetina y descubrimiento de los elementos de respuesta a hipoxia (HRE) en la región promotora y posteriormente al factor transcripcional inducible por hipoxia tipo 1 (HIF-1). Este factor consta de dos subunidades: HIF-1α, sensible al oxígeno, y HIF-1β, expresada constitutivamente. HIF1 activa la transcripción de genes que codifican enzimas, transportadores y proteínas mitocondriales que disminuyen la utilización de oxígeno al cambiar el metabolismo oxidativo al metabolismo glicolítico y además aquellos involucrados en la angiogénesis y diferenciación celular. Las investigaciones paralelas en la enfermedad von Hippel-Lindau (VHL), un desorden autosómico dominante, permitieron descubrir el mecanismo de degradación de HIF1 en condiciones de normoxia y como se estabiliza bajo hipoxia. El impacto de HIF en clínica radica en el establecimiento de nuevas dianas terapéuticas para combatir la anemia y diversas enfermedades cardiovasculares. HIF promueve la angiogénesis a través de la expresión del factor de crecimiento vascular endotelial (VEGF), agente cardioprotector con potencial para tratar la isquemia/reperfusión, hipertrofia patológica e insuficiencia cardíaca.

ABSTRACT: The Nobel Prize in Physiology-Medicine was awarded to Drs. Gregg Semenza, William Kaelin and Sir Peter Ratcliffe for their research in the molecular machinery that regulates the expression of genes sensitive to the change in oxygen levels. The synthesis of erythropoietin induced by the decrease levels of oxygen in the blood led to investigate the promoter of the erythropoietin gene where the so-called hypoxia response elements (HRE) were described. Semenza et al. described a protein that binds to HREs and called it hypoxia-inducible transcriptional factor (HIF) that regulates gene expression among those involved in angiogenesis, cell differentiation and glycolytic enzymes. HIF presents two oxygen-sensitive subunits HIF-1α and HIF-1β constitutively expressed. In parallel, Kaelin et al. investigated von Hippel-Lindau disease (VHL), an autosomal dominant disorder, discovering a mutation of this protein generated a behavior similar to hypoxia. The impact of HIF-1α lies in the search for new strategies such as hydrolase inhibitors to combat prevalent diseases, including anemia and cardiovascular diseases These compounds promote the expression of vascular endothelial growth factor (VEGF), a cardioprotective agent with potential use in pre- and post-conditioning therapy, cardiac hypertrophy and heart failure.

Regulatory Role of Hypoxia Inducible Factor in the Biological Behavior of Nucleus Pulposus Cells

Intervertebral disc (IVD) degeneration is implicated as a major cause of low back pain. The alternated phenotypes, reduced cell survival, decreased metabolic activity, loss of matrix production and dystrophic mineralization of nucleus pulposus (NP) cells may be key contributors to progressive IVD degeneration. IVD is the largest avascular structure in the body, characterized by low oxygen tension in vivo. Hypoxia-inducible factor (HIF) is a master transcription factor that is induced upon hypoxia and directs coordinated cellular responses to hypoxic environments. This review summarizes relevant studies concerning the involvement of HIF in the regulation of biological behaviors of NP cells. We describe current data on the expression of HIF in NP cells and further discuss the various roles that HIF plays in the regulation of the phenotype, survival, metabolism, matrix production and dystrophic mineralization of NP cells. Here, we conclude that HIF may be a promising target for the prevention and treatment of IVD degeneration.

Hypoxia and angiogenesis: regulation of hypoxia-inducible factors via novel binding factors

The mechanisms that regulate angiogenesis in hypoxia or hypoxic microenvironment are modulated by several pro- and antiangiogenic factors. Hypoxia-inducible factors (HIFs) have been established as the basic and major inducers of angiogenesis, but understanding the role of interacting proteins is becoming increasingly important to elucidate the angiogenic processes of a hypoxic response. In particular, with regard to wound healing and the novel therapies for vascular disorders such as ischemic brain and heart attack, it is essential to gain insights in the formation and regulation of HIF transcriptional machineries related to angiogenesis. Further, identification of alternative ways of inhibiting tumor growth by disrupting the growth-triggering mechanisms of increasing vascular supply via angiogenesis depends on the knowledge of how tumor cells develop their own vasculature. Here, we review our findings on the interactions of basic HIFs, HIF-1alpha and HIF-2alpha, with their regulatory binding proteins, histone deacetylase 7 (HDAC7) and translation initiation factor 6 (Int6), respectively. The present results and discussion revealed new regulatory interactions of HIF-related mechanisms.