Raghavan Raju

Sirtuin regulation in aging and injury

Sirtuins or Sir2 family of proteins are a class of NAD(+) dependent protein deacetylases which are evolutionarily conserved from bacteria to humans. Some sirtuins also exhibit mono-ADP ribosyl transferase, demalonylation and desuccinylation activities. Originally identified in the yeast, these proteins regulate key cellular processes like cell cycle, apoptosis, metabolic regulation and inflammation. Humans encode seven sirtuin isoforms SIRT1-SIRT7 with varying intracellular distribution. Apart from their classic role as histone deacetylases regulating transcription, a number of cytoplasmic and mitochondrial targets of sirtuins have also been identified. Sirtuins have been implicated in longevity and accumulating evidence indicate their role in a spectrum of diseases like cancer, diabetes, obesity and neurodegenerative diseases. A number of studies have reported profound changes in SIRT1 expression and activity linked to mitochondrial functional alterations following hypoxic-ischemic conditions and following reoxygenation injury. The SIRT1 mediated deacetylation of targets such as PGC-1α, FOXO3, p53 and NF-κb has profound effect on mitochondrial function, apoptosis and inflammation. These biological processes and functions are critical in life-span determination and outcome following injury. Aging is reported to be characterized by declining SIRT1 activity, and its increased expression or activation demonstrated prolonged life-span in lower forms of animals. A pseudohypoxic state due to declining NAD(+) has also been implicated in aging. In this review we provide an overview of studies on the role of sirtuins in aging and injury.

Mitochondrial function in hypoxic ischemic injury and influence of aging.

HIGHLIGHTSNuclear‐mitochondrial cross talk is important in maintaining mitochondrial structure and function.Metabolic master regulators, c‐MYC and HIF‐1, modulate mitochondrial function and cell fate following hypoxic/ischemic injury.Aging exacerbates hypoxic/ischemic injury and mitochondrial functional impairment.AMPK‐SIRT1 mediated energy sensing is important in energy homeostasis following hypoxic/ischemic injury.Post‐translational changes are critical in the control of the glycolytic‐mitochondrial energy axis in response to hypoxic/ischemic conditions. ABSTRACT Mitochondria are a major target in hypoxic/ischemic injury. Mitochondrial impairment increases with age leading to dysregulation of molecular pathways linked to mitochondria. The perturbation of mitochondrial homeostasis and cellular energetics worsens outcome following hypoxic‐ischemic insults in elderly individuals. In response to acute injury conditions, cellular machinery relies on rapid adaptations by modulating posttranslational modifications. Therefore, post‐translational regulation of molecular mediators such as hypoxia‐inducible factor 1&agr; (HIF‐1&agr;), peroxisome proliferator‐activated receptor &ggr; coactivator &agr; (PGC‐1&agr;), c‐MYC, SIRT1 and AMPK play a critical role in the control of the glycolytic‐mitochondrial energy axis in response to hypoxic‐ischemic conditions. The deficiency of oxygen and nutrients leads to decreased energetic reliance on mitochondria, promoting glycolysis. The combination of pseudohypoxia, declining autophagy, and dysregulation of stress responses with aging adds to impaired host response to hypoxic‐ischemic injury. Furthermore, intermitochondrial signal propagation and tissue wide oscillations in mitochondrial metabolism in response to oxidative stress are emerging as vital to cellular energetics. Recently reported intercellular transport of mitochondria through tunneling nanotubes also play a role in the response to and treatments for ischemic injury. In this review we attempt to provide an overview of some of the molecular mechanisms and potential therapies involved in the alteration of cellular energetics with aging and injury with a neurobiological perspective.

Aging and Injury: Alterations in Cellular Energetics and Organ Function

Aging is characterized by increased oxidative stress, heightened inflammatory response, accelerated cellular senescence and progressive organ dysfunction. The homeostatic imbalance with aging significantly alters cellular responses to injury. Though it is unclear whether cellular energetic imbalance is a cause or effect of the aging process, preservation of mitochondrial function has been reported to be important in organ function restoration following severe injury. Unintentional injuries are ranked among the top 10 causes of death in adults of both sexes, 65 years and older. Aging associated decline in mitochondrial function has been shown to enhance the vulnerability of heart, lung, liver and kidney to ischemia/reperfusion injury. Studies have identified alterations in the level or activity of factors such as SIRT1, PGC-1α, HIF-1α and c-MYC involved in key regulatory processes in the maintenance of mitochondrial structural integrity, biogenesis and function. Studies using experimental models of hemorrhagic injury and burn have demonstrated significant influence of aging in metabolic regulation and organ function. Understanding the age-associated molecular mechanisms regulating mitochondrial dysfunction following injury is important towards identifying novel targets and therapeutic strategies to improve the outcome after injury in the elderly.

Resveratrol Suppresses Expression of VEGF by Human Retinal Pigment Epithelial Cells: Potential Nutraceutical for Age-related Macular Degeneration

Age-related macular degeneration (AMD) is a sight threating retinal eye disease that affects millions of aging individuals world-wide. Choroid-retinal pigment epithelium (RPE)-neuroretina axis in the posterior compartment of the eye is the primary site of AMD pathology. There are compelling evidence to indicate association of vascular endothelial growth factors (VEGF) to AMD. Here, we report the inhibitory actions of resveratrol (RSV) on inflammatory cytokine, TGF-β and hypoxia induced VEGF secretion by human retinal pigment epithelial cells (HRPE). HRPE cultures prepared from aged human donor eyes were used for the studies in this report. HRPE secreted both VEGF-A and VEGF-C in small quantities constitutively. Stimulation with a mixture of inflammatory cytokines (IFN-γ, TNF-α, IL-1β), significantly increased the secretion of both VEGF-A and VEGF-C. RSV, in a dose dependent (10-50 uM) manner, suppressed VEGF-A and VEGF-C secretion induced by inflammatory cytokines significantly. RT-PCR analysis indicated that effects of RSV on VEGF secretion were possibly due to decreased mRNA levels. TGF-β and cobalt chloride (hypoxia mimic) also upregulated HRPE cell production of VEGF-A, and this was inhibited by RSV. In contrast, RSV had no effect on anti-angiogenic molecules, endostatin and pigment epithelial derived factor secretion. Studies using an in vitro scratch assay revealed that wound closure was also inhibited by RSV. These results demonstrate that RSV can suppress VEGF secretion induced by inflammatory cytokines, TGF-β and hypoxia. Under pathological conditions, over expression of VEGF is known to worsen AMD. Therefore, RSV may be useful as nutraceutical in controlling pathological choroidal neovascularization processes in AMD.

Resveratrol Improves Survival and Prolongs Life Following Hemorrhagic Shock

Resveratrol has been shown to potentiate mitochondrial function and extend longevity; however, there is no evidence to support whether resveratrol can improve survival or prolong life following hemorrhagic shock. We sought to determine whether (a) resveratrol can improve survival following hemorrhage and resuscitation and (b) prolong life in the absence of resuscitation. Using a hemorrhagic injury (HI) model in the rat, we describe for the first time that the naturally occurring small molecule, resveratrol, may be an effective adjunct to resuscitation fluid. In a series of three sets of experiments we show that resveratrol administration during resuscitation improves survival following HI (p < 0.05), resveratrol and its synthetic mimic SRT1720 can significantly prolong life in the absence of resuscitation fluid (<30 min versus up to 4 h; p < 0.05), and resveratrol as well as SRT1720 restores left ventricular function following HI. We also found significant changes in the expression level of mitochondria-related transcription factors Ppar-α and Tfam, as well as Pgc-1α in the left ventricular tissues of rats subjected to HI and treated with resveratrol. The results indicate that resveratrol is a strong candidate adjunct to resuscitation following severe hemorrhage.

Alteration of cytokine profile following hemorrhagic shock

Hemorrhage is one of the leading causes of death in patients with trauma. We recently demonstrated that resveratrol can improve cardiac function and prolong life following severe hemorrhagic injury (HI) in a rat model. The present work is focused on determining changes in NF-κB dependent gene expression in the heart and the systemic cytokine milieu following HI and the effect of resveratrol treatment. The results indicate an increase in phosphorylated NF-κB in the heart with a concomitant increase in the expression of NF-κB dependent genes following HI. There was also a significant increase of systemic cytokine levels, both pro and anti-inflammatory, following HI and resolution when treated with resveratrol. This study demonstrates the potential role NF-κB has in the physiological response to HI and the effectiveness of resveratrol in reducing immune activation.

Mitochondrial targeting by dichloroacetate improves outcome following hemorrhagic shock

Hemorrhagic shock is a leading cause of death in people under the age of 45 and accounts for almost half of trauma-related deaths. In order to develop a treatment strategy based on potentiating mitochondrial function, we investigated the effect of the orphan drug dichloroacetate (DCA) on survival in an animal model of hemorrhagic shock in the absence of fluid resuscitation. Hemorrhagic shock was induced in rats by withdrawing 60% of the blood volume and maintaining a hypotensive state. The studies demonstrated prolonged survival of rats subjected to hemorrhagic injury (HI) when treated with DCA. In separate experiments, using a fluid resuscitation model we studied mitochondrial functional alterations and changes in metabolic networks connected to mitochondria following HI and treatment with DCA. DCA treatment restored cardiac mitochondrial membrane potential and tissue ATP in the rats following HI. Treatment with DCA resulted in normalization of several metabolic and molecular parameters including plasma lactate and p-AMPK/AMPK, as well as Ach-mediated vascular relaxation. In conclusion we demonstrate that DCA can be successfully used in the treatment of hemorrhagic shock in the absence of fluid resuscitation; therefore DCA may be a good candidate in prolonged field care following severe blood loss.

Kidney-targeted inhibition of protein kinase C-α ameliorates nephrotoxic nephritis with restoration of mitochondrial dysfunction

To investigate the role of protein kinase C-α (PKC-α) in glomerulonephritis, the capacity of PKC-α inhibition to reverse the course of established nephrotoxic nephritis (NTN) was evaluated. Nephritis was induced by a single injection of nephrotoxic serum and after its onset, a PKC-α inhibitor was administered either systemically or by targeted glomerular delivery. By day seven, all mice with NTN had severe nephritis, whereas mice that received PKC-α inhibitors in either form had minimal evidence of disease. To further understand the underlying mechanism, label-free shotgun proteomic analysis of the kidney cortexes were performed, using quantitative mass spectrometry. Ingenuity pathway analysis revealed 157 differentially expressed proteins and mitochondrial dysfunction as the most modulated pathway. Functional protein groups most affected by NTN were mitochondrial proteins associated with respiratory processes. These proteins were down-regulated in the mice with NTN, while their expression was restored with PKC-α inhibition. This suggests a role for proteins that regulate oxidative phosphorylation in recovery. In cultured glomerular endothelial cells, nephrotoxic serum caused a decrease in mitochondrial respiration and membrane potential, mitochondrial morphologic changes and an increase in glycolytic lactic acid production; all normalized by PKC-α inhibition. Thus, PKC-α has a critical role in NTN progression, and the results implicate mitochondrial processes through restoring oxidative phosphorylation, as an essential mechanism underlying recovery. Importantly, our study provides additional support for targeted therapy to glomeruli to reverse the course of progressive disease.

Effect of plasma-derived extracellular vesicles on erythrocyte deformability in polymicrobial sepsis

&NA; Sepsis affects microcirculation and tissue perfusion leading to tissue hypoxia and multiple organ dysfunction. Red blood cells (RBCs; erythrocytes) are typically biconcave in shape, transport hemoglobin‐bound oxygen and are reversibly deformable facilitating trafficking through capillaries. Decreased deformability of RBCs adversely affects tissue oxygenation. The purpose of this project was to determine RBC deformability in a murine model of polymicrobial sepsis by a method that utilizes laser diffraction and microfluidics, and to identify the causative factors in the plasma that may contribute to loss in RBC deformability. Blood samples from mice subjected to cecal ligation and puncture (CLP) model of sepsis were used. RBC deformability was tested using Rheoscan‐AnD 300 under shear stress range of 0–20 Pascal (Pa) that depicts the common rheological behavior of RBCs flowing through blood vessels ranging from major vessels to capillaries. Normal RBCs were treated with plasma‐derived extracellular vesicles (EVs) and their effect on RBC deformability was also tested. The experiments demonstrated a significant decrease in RBC deformability following sepsis. RBC deformability recovered in sham‐operated animals by the third day, whereas animals with sepsis continued to show decreased levels of deformability. EVs isolated from the plasma of animals from the sepsis group significantly decreased deformability of RBCs ex vivo. Analysis of miRNA cargo in EVs showed distinct molecular profiles for sham‐operated and sepsis‐induced mice. In summary, sepsis induced a decrease in RBC deformability and the acquired rigidity may have adverse effect on microcirculation, tissue perfusion, and organ function.