Ziv Radisavljevic

Oxidative Modification of Mitochondrial Integrity and Nerve Fiber Density in the Ischemic Overactive Bladder

PURPOSE To our knowledge the mechanism of neurodegeneration in the overactive bladder remains unknown. We examined mitochondrial integrity and searched for markers of oxidative neural injury in the ischemic overactive bladder. MATERIALS AND METHODS A rabbit model of overactive bladder was developed by inducing moderate pelvic ischemia. After 16 weeks cystometrograms and blood flow recordings from overactive bladders were compared with those in age matched controls. Bladder tissues were processed to assess oxidative products, oxidative stress sensitive genes and nerve fiber density using enzyme immunoassay, quantitative real-time polymerase chain reaction and immunohistochemical staining, respectively. Tissue ultrastructure was examined by transmission electron microscopy. RESULTS Ischemia increased spontaneous bladder contractions and led to cyclic ischemia-reperfusion. Tissue levels of oxidative and nitrosative products, and oxidative stress sensitive genes encoding superoxide dismutase and aldose reductase were up-regulated in the overactive bladder. Transmission electron microscopy of overactive bladder tissues showed mitochondria with distinctive morphological features, characterized by swollen membranes, decreased granules, a total loss of granules and sporadic membrane damage. These changes were associated with sporadic loss of epithelial mucosal membrane, twisted smooth muscle cells, diffused vacuolization and marked neurodegeneration. CONCLUSIONS Our findings suggest free radical mediated ultrastructural damage and neurodegeneration in the overactive bladder. Overactivity associated mitochondrial stress may have a central role in epithelial damage, smooth muscle cell injury and neurodegeneration. Superoxide dismutase and aldose reductase up-regulation in the overactive bladder imply intrinsic defensive reaction against free radicals that apparently fails to prevent oxidative damage and neurodegeneration. Therapeutic strategies targeting basic mitochondrial processes such as energy metabolism or free radical generation may help better manage wall degeneration and neuropathy in the overactive bladder.

Dietary antioxidants improve arteriogenic erectile dysfunction

Most cases of erectile dysfunction (ED) are associated with oxidative stress risk factors such as diabetes mellitus, smoking, hypercholesterolaemia and hypertension. Our goal was to search for markers of oxidative stress in arteriogenic ED and examine the protective role of dietary antioxidants. Atherosclerosis-induced ED was developed in rabbits by balloon de-endothelialization of the iliac arteries. Ballooned and age-matched control animals were assigned into subgroups receiving pomegranate extract antioxidants in drinking water or tap water as placebo. After 8 weeks, penile blood flow and erectile activity were recorded. Erectile tissue relaxation, oxidative products, oxidative stress-responsive genes and structure were examined using organ bath, enzyme immunoassay, quantitative real-time polymerase chain reaction and transmission electron microscopy, respectively. Arterial ballooning caused diffused atherosclerosis, decreased intracavernosal blood flow and led to ED. Impairment of endothelium-dependent relaxation, diffused fibrosis, increased oxidative products, upregulation of superoxide dismutase (SOD) and aldose reductase (AR) gene expression, mitochondrial and endothelial structural damage and increased caveolae were evident in erectile tissues from atherosclerotic animals receiving placebo. Upregulation of antioxidant enzymes SOD and AR failed to protect ischaemic erectile tissue from oxidative injury. Pomegranate extract significantly improved intracavernosal blood flow, erectile activity, smooth muscle relaxation and fibrosis of the atherosclerotic group in comparison with the atherosclerotic group receiving placebo, but did not normalize them to the age-matched control levels. Pomegranate extract appeared more effective in diminishing oxidative products, preventing SOD and AR gene upregulation, and protecting mitochondrial, endothelial and caveolae structural integrity of the atherosclerotic group. Our data suggest the presence of oxidative stress in ED and a more efficient action of antioxidants on molecular and ultrastructural alterations than on distinct functional deficit and structural damage in the ischaemic penis.

Molecular Reactions and Ultrastructural Damage in the Chronically Ischemic Bladder

PURPOSE Clinical and basic research data suggest that pelvic ischemia may contribute to bladder overactivity. We characterized the molecular and ultrastructural reactions of the chronically ischemic bladder. MATERIALS AND METHOD A model of pelvic ischemia was developed by creating iliohypogastric/pudendal arterial atherosclerosis in rabbits. At 12 weeks conscious urinary frequency was examined, bladder blood flow was recorded and cystometrograms were done using general anesthesia. Bladder tissue was processed for molecular and ultrastructural analysis using quantitative real-time polymerase chain reaction, Western blot and transmission electron microscopy. RESULTS Conscious urinary frequency and the frequency of spontaneous bladder contractions significantly increased in animals with pelvic ischemia. Bladder ischemia up-regulated the gene and protein expression of hypoxia inducible factor-1α, transforming growth factor-β and nerve growth factor B. Vascular endothelial growth factor gene expression also increased but protein levels were unchanged. Transmission electron microscopy of ischemic bladder samples showed swollen mitochondria with degraded granules, thickened epithelium, deformed muscle fascicles, collagen deposition and impaired microvasculature with thickened intima and disrupted endothelial cell junctions. Degenerating axonal and Schwann cell profiles, and myelin sheath splitting around axons and Schwann cells were evident in ischemic bladders. CONCLUSIONS Interrupting pelvic blood flow resulted in an ischemic overactive bladder and significant increase in conscious urinary frequency. Molecular responses involving hypoxia inducible factor, transforming growth factor-β, vascular endothelial growth factor and nerve growth factor were associated with mitochondrial injury, fibrosis, microvasculature damage and neurodegeneration. Ischemia may have a key role in bladder overactivity and lower urinary tract symptoms.

TOR kinase and ran are downstream from PI3K/Akt in H2O2-induced mitosis

Hydrogen peroxide (H2O2) activates signaling cascades essential for cell proliferation via phosphatidylinositol‐3‐kinase (PI3K) and Akt. Here we show that induction of mitogenic signaling by H2O2 activates sequentially PI3K, Akt, mammalian target of rapamycin (mTOR), and Ran protein. Akt activation is followed by signaling through the mTOR kinase and upregulation of Ran in primary type II pneumocytes, a cell type implicated in the development of lung adenocarcinoma. Pretreatment of the cells with wortmannin, a specific inhibitor of PI3K, or rapamycin, a specific inhibitor of mTOR kinase, prevented H2O2‐increased mitosis. H2O2‐induced Akt ser‐473 phosphorylation and upregulation of Ran protein were prevented by wortmannin but not by rapamycin, indicating that PI3K is upstream of Akt and mTOR is downstream from Akt. Overexpression of myr‐Akt or Ran‐wt in type II pneumocytes increased Akt ser‐473 phosphorylation and mitosis in a catalase‐dependent manner, indicating that H2O2 is essential for Akt and Ran signaling. These results indicate that H2O2‐induced mitogenic signaling in primary type II pneumocytes is mediated by PI3K, Akt, mTOR‐kinase, and Ran protein.

Inactivated tumor suppressor Rb by nitric oxide promotes mitosis in human breast cancer cells

Inactivation of tumor suppressor protein retinoblastoma (Rb) is important mechanism for the G1/S transition during cell cycle progression. Human breast cancer cells T47D release great amount of nitric oxide (NO), but its relation to tumor suppressor Rb is unknown. In this study, it is shown that NO induces phosphorylation and inactivation of Rb tumor suppressor protein, increasing G2/M phase and cell proliferation of breast cancer cells T47D. NO did not induce changes in p53 ser‐15 phosphorylation, the most phosphorylated site of p53 during its activation. These data indicate that NO induces cell proliferation through the Rb pathway. NO phosphorylates and inactivates tumor suppressor protein Rb inducing mitosis by the p53 independent pathway in breast cancer cell.

AKT as locus of cancer multidrug resistance and fragility.

Complexity and robustness of cancer hypoxic microenvironment are supported by the robust signaling networks of autocrine and paracrine elements creating powerful interactome for multidrug resistance. These elements generate a positive feedback loops responsible for the extreme robustness and multidrug resistance in solid cancer, leukemia, myeloma, and lymphoma. Phosphorylated AKT is a cancer multidrug resistance locus. Targeting that locus by oxidant/antioxidant balance modulation, positive feedback loops are converted into negative feedback loops, leading to disappearance of multidrug resistance. This is a new principle for targeting cancer multidrug resistance by the locus chemotherapy inducing a phenomenon of loops conversion. J. Cell. Physiol. 228: 671–674, 2013.

Loss of bladder smooth muscle caveolae in the aging bladder

Caveolae are specialized regions of the cell membrane that modulate signal transduction and alterations in these structures affect bladder smooth muscle (BSM) contraction. Since bladder dysfunctions are common in the elderly, we evaluated the effect of aging on the morphology of caveolae and caveolin protein expression in BSM.

AKT as locus of cancer angiogenic robustness and fragility.

Angiogenesis get full robustness in metastatic cancer, relapsed leukemia or lymphoma when complex positive feedback loop signaling systems become integrative. A cancer hypoxic microenvironment generates positive loops inducing formation of the vascular functional shunts. AKT is an upstream angiogenic locus of integrative robustness and fragility activated by the positive loops. AKT controls two downstream nodes the mTOR and NOS in nodal organization of the signaling genes. AKT phosphorylation is regulated by a balance of an oxidant/antioxidant. Targeting AKT locus represents new principle to control integrative angiogenic robustness by the locus chemotherapy. J. Cell. Physiol. 228: 21–24, 2013.

Effects of ischemia on tachykinin-containing nerves and neurokinin receptors in the rabbit bladder.

OBJECTIVES Our previous studies showed marked changes in efferent nerve structure and reactivity in the ischemic bladder. The goal of this study was to examine the effects of bladder ischemia on tachykinin (TK) containing sensory nerves and neurokinin receptors (NKR) in a rabbit model. METHODS We recorded bladder blood flow and spontaneous contractions in treated animals at week 8 after the induction of iliac arteries atherosclerosis and in age-matched controls. Bladder tissues were processed for studies of isometric smooth muscle tension in the organ bath, NK2R gene expression using quantitative real-time polymerase chain reaction (PCR), immunohistochemical staining of TK containing nerves and epithelial TK expression, and transmission electron microscopy. RESULTS Atherosclerosis-induced ischemia significantly increased the frequency of spontaneous bladder contractions in vivo. Electrical field stimulation (EFS)-induced smooth muscle contractions were significantly greater in the ischemic tissues. Inhibition of NK1R diminished contractions to low-frequency EFS in control tissues while having no significant effect on the ischemic tissues. In contrast, NK2R inhibition significantly decreased contractions to both low- and high-frequency EFS in the ischemic tissues. Inhibition of NK3R had no significant effect on EFS-induced contractions. Real-time PCR showed a significant increase in NK2R gene expression in the ischemic bladder. The number of TK immunopositive nerves and epithelial TK immunoreactivity were significantly greater in the ischemic bladder. These alterations were associated with marked ultrastructural reactions to bladder ischemia. CONCLUSIONS Alterations of NK2R reactivity and gene expression, increased number of TK immunopositive nerves, and greater epithelial TK immunoreactivity may imply activated bladder afferents to signal ischemic insult.

Locus of fragility in robust breast cancer system.

Functional heterogeneous redundancy of breast cancer makes this tumor to be robust. Signaling mechanisms which control cancer responses are crucial for controlling robustness. Identification of locus of fragility in cancer represents basic mechanism to target robustness. The goal of this prospect is to present locus of fragility in breast cancer robust system, and how disruption of this locus induces failure of robustness. My recent research show, that locus of fragility in breast cancer cells is suppression of nitric oxide (NO). When it was targeted, dynamics of cancer to generate robustness failed that it blocked cancer cell proliferation dependent on the NO/Rb pathway, blocked cell migration and angiogenesis dependent on the VEGF/PI3K/AKT/NO/ICAM‐1 pathway, and induced breast cancer cell apoptosis through the NO/ROCK/FOXO3a signaling pathway. This tiny and trivial perturbation in breast cancer cells such as suppression of NO represents locus of fragility (weakness) and new approach for breast cancer chemotherapy.