Magomed Khaidakov - Academia.edu (original) (raw)
Papers by Magomed Khaidakov
Human Mutation, 1997
The relative contribution of both genetic and environmental factors to spontaneous mutation frequ... more The relative contribution of both genetic and environmental factors to spontaneous mutation frequency in humans is unknown. We have investigated the contribution of genetic factors to this phenomenon by determining the in vivo mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in circulating T-lymphocytes obtained from pairs of monozygotic twins. hprt mutant frequencies were determined three times over fourteen days in six sets of monozygotic male twins (mean age 30) taking part in a Russian Space Program inclined bed rest experiment. Blood samples were obtained prior to, during, and immediately following the experiment. Mononuclear cells were separated, frozen, and flown to Canada for analysis using the hprt T-lymphocyte clonal assay. There is no evidence within this data set to demonstrate that the period of inclined bed rest to simulate the effects of weightlessness had any effect on the observed mutant frequency. However, the average mutant frequency for the six sets of Russian twins was found to be three times higher than that of Western counterparts. More surprisingly, the spontaneous mutant frequency of monozygotic twins was found to be much more similar within pairs than between pairs of twins. These data suggest that the contribution of genetics in the determination of mutation frequency is substantial. However, whether high concordance within twin pairs reflects shared environmental experience as well as common genetic factors is not entirely clear. More data will be required to distinguish genetic from environmental factors and to determine the degree to which mutant frequency is genetically determined.
Mechanisms of Ageing and Development, 2006
Mitochondria have long been suspected to be among the leading determinants of aging due to their ... more Mitochondria have long been suspected to be among the leading determinants of aging due to their functional importance and accelerated deterioration caused by accumulation of mutations in the mitochondrial DNA. Direct repeats are known to contribute to deletion formation in mtDNA and are a powerful source of reactive oxygen species (ROS)-independent mutagenesis. To evaluate the potential importance of homology-based deletion formation, we have analyzed the association between direct repeats in the mtDNA sequence and the lifespans of 65 mammalian species. Here, we report a significant negative correlation between the mutagenic potential of direct repeats and the mammalian lifespan, which is especially evident in closely related species.
Journal of the American College of Cardiology, 2011
Background: Activation of LOX-1, a lectin-like ox-LDL receptor-1, exerts a significant role in co... more Background: Activation of LOX-1, a lectin-like ox-LDL receptor-1, exerts a significant role in collagen formation. We hypothesized that LOX-1 deletion may inhibit inflammatory/oxidative stress signals and reduce collagen accumulation, and attenuate cardiac remodeling after chronic ischemia.
Experimental Gerontology, 2005
This study analyzed the incidence of point mutations in mitochondrial DNA of brain and muscle tis... more This study analyzed the incidence of point mutations in mitochondrial DNA of brain and muscle tissues from young (6-month) and old (24-month) male F344 rats. Coding sequence mutations in subunit 5 of the NADH dehydrogenase gene were detected after high-fidelity PCR amplification and cloning by denaturing gradient gel electrophoresis (DGGE) assay followed by sequencing of detected mutants. In total, almost a thousand individual clones were analyzed both in brain and muscle samples. On average, mtDNA from brain tissue showed a 66% increase with age in mutation frequencies (2.3G1.9 vs. 3.8G4.5!10 K4 mutations/bp, meanGSD), which failed to reach statistical significance (pZ0.45). Muscle tissues yielded substantially fewer mutants with average mutant frequencies for both young and old rats almost 10 times lower than the corresponding values in the brain tissue (0.3G0.4 and 0.5G0.6!10 K4 , respectively). The difference in mutation accumulation between muscle and brain was highly significant in both the younger group (Chi-squaredZ9.7, p%0.01) and in older animals (Chi-squaredZ10.9, p%0.001). Molecular analysis of the mutated sequences revealed that almost half were identical sequences recurring in different samples and tissues. Our findings indicate that the process of mutation accumulation in mitochondria begins in the germ-line and/or during earlier stages of life, contributing up to half of the total mutational burden, whereas de novo spontaneous formation of point mutations in adulthood is far less than was anticipated. q 2005 Elsevier Inc. All rights reserved.
Accumulating evidence suggests that the aging process is, in part, driven by accumulation of larg... more Accumulating evidence suggests that the aging process is, in part, driven by accumulation of large deletions in mitochondrial DNA (mtDNA). Here, I present a hypothesis that significant variations in lifespans can be explained by species-specific mtDNA sequence features that cause a shift in the mode of mtDNA replication and thus preclude the formation of large deletions.
PLoS ONE, 2012
Dyslipidemia and obesity are primary risk factors for the development of atherosclerosis and are ... more Dyslipidemia and obesity are primary risk factors for the development of atherosclerosis and are also epidemiologically linked to increased susceptibility to a variety of cancers including breast cancer. One of the prominent features of dyslipidemia is enhanced production of oxidized LDL (ox-LDL), which has been shown to be implicated in key steps of atherogenesis including inflammatory signaling and proliferation of vascular cells. In this study we analyzed the effects of ox-LDL in human mammary epithelial cells (MCF10A). MCF10A cells avidly internalized dil-ox-LDL and exhibited increased proliferative response to ox-LDL within the range of 1-50 mg/ml in a dose-dependent manner. Treatment of cells with 20 mg/ml ox-LDL for 2 and 12 hours was associated with upregulation of LOX-1 and CD36 scavenger receptors while MSR1 and CXLC16 receptors did not change. Ox-LDL-treated cells displayed significant upregulation of NADPH oxidases (subunits P22 phox and P47 phox ), lipoxygenases-12 and -15, and cytoplasmic, but not mitochondrial, SOD. Ox-LDL also triggered phosphorylation of IkBa coupled with nuclear translocation of NF-kB and stimulated p44/42 MAPK, PI3K and Akt while intracellular PTEN (PI3K/Akt pathway inhibitor and target of miR-21) declined. Quantitative PCR revealed increased expression of hsa-miR-21 in ox-LDL treated cells coupled with inhibition of miR-21 target genes. Further, transfection of MCF10A cells with miR-21 inhibitor prevented ox-LDL mediated stimulation of PI3K and Akt. We conclude that, similarly to vascular cells, mammary epithelial cells respond to ox-LDL by upregulation of proliferative and pro-inflammatory signaling. We also report for the first time that part of ox-LDL triggered reactions in MCF10A cells is mediated by oncogenic hsa-miR-21 through inhibition of its target gene PTEN and consequent activation of PI3K/Akt pathway.
Biochemical Basis and Therapeutic Implications of Angiogenesis, 2013
PLoS ONE, 2011
Numerous studies have described the process of senescence associated with accumulation of oxidati... more Numerous studies have described the process of senescence associated with accumulation of oxidative damage, mutations and decline in proliferative potential. Although the changes observed in senescent cells are likely to result in significant phenotypic alterations, the studies on consequences of endothelial senescence, especially in relation to aging-associated diseases, are scarce. We have analyzed effects of senescence on the functions of endothelial cells relevant to the development of atherosclerosis including angiogenesis, adhesion, apoptosis and inflammation. In the course of progressing through the passages, human umbilical vein endothelial cells (HUVECs) displayed significant increase in size (+36% passage 12 vs. passage 4 , p,0.001) and reduction in both basal and VEGF-stimulated tube formation. The analysis of a scavenger receptor LOX-1, a key molecule implicated in atherogenesis, revealed a significant decline of its message (mRNA) and protein content in senescent endothelial cells (233%) and in aortas of 50 wk (vs. 5 wk) old mice (all p,0.01). These effects were accompanied by a marked reduction of the basal expression of VCAM-1 and ICAM-1. Compared to early cultures, late passage HUVECs also exhibited nuclear translocation of NF-kB (p65) and reciprocal shifts in BAX and BCL2 protein content resulting in almost 2-fold increase in BAX/BCL2 ratio and 3-fold increase in apoptotic response to TNFa exposure (p,0.04). These changes in senescent endothelial cells are suggestive of aberrant responses to physiological stimuli resulting in a less permissive environment for tissue remodeling and progression of diseases requiring angiogenesis and cell adhesion in elderly, possibly, mediated by LOX-1.
Cardiovascular Drugs and Therapy, 2011
... Oncogene. 2004;23:636578. 3. Lorincz AM, Sukumar S. Molecular links between obesity and brea... more ... Oncogene. 2004;23:636578. 3. Lorincz AM, Sukumar S. Molecular links between obesity and breast cancer. Endocr Relat Canc. 2006;13:27992. 4. Mehta JL. ... Physiol Pharma-col. 2000;51:88396. 15. Roberts CK, Barnard RJ, Sindhu RK, Jurczak M, Ehdaie A, Vaziri ND. ...
Cardiovascular research, 2014
Journal of cardiovascular pharmacology and therapeutics, 2011
Hypoxia-reoxygenation (HR) is a major driver for angiogenesis in atherosclerotic plaques and tumo... more Hypoxia-reoxygenation (HR) is a major driver for angiogenesis in atherosclerotic plaques and tumors. Angiogenesis is a multistep process requiring stimulation of proliferation and migration of endothelial cells in response to a number of growth factors, including transforming growth factor (TGFβ1). Aspirin (acetylsalicylic acid) has been shown to reduce atherosclerosis-related events as well as development of certain tumors. We examined the role of aspirin in HR-mediated angiogenesis from human umbilical vein endothelial cells (HUVECs). We found that aspirin (0.5 and 1 mmol/L) markedly (by about 30%, P < .01) reduced HR-mediated tube formation. Next, we studied changes in TGFβ1 and its type 1 receptor (TGFβ-R1) as well as in the transcription of p53 and p21 during HR-mediated angiogenesis. Hypoxia-reoxygenation modestly increased TGFβ1 and decreased its type 1 receptor (TGFβ-R1) transcription (both P-NS) and reduced the transcription of p53 and p21 (P < .05). Treatment of HUVE...
Molecular cancer, 2005
Several studies of tumors have revealed substantial numbers of clonally expanded somatic mutation... more Several studies of tumors have revealed substantial numbers of clonally expanded somatic mutations in mitochondrial DNA (mtDNA), not observed in adjacent intact tissues. These findings were interpreted as indicating the involvement of mtDNA mutations in tumorigenesis. Such comparisons, however, ignore an important confounding factor: the monoclonal origin of tumors as opposed to the highly polyclonal nature of normal tissues. Analysis of recently published data on the incidence of somatic mutations in nontumor monoclonal cells suggests that, contrary to the prevailing view, the process of tumorigenesis may be accompanied by active selection against detrimental mtDNA mutations.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2001
Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals prov... more Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals provide end points that can be used for assessing the antimutagenic or anticarcinogenic properties of putative chemopreventive compounds and for predicting their protective efficacy in humans. In this study, we investigated the ability of the dietary antioxidant Vitamins C, E, beta-carotene and the mineral selenium to inhibit the mutant frequency (MF) induced by treatment of rats with 7,12-dimethylbenz[a]anthracene (DMBA), a mammary carcinogen and bleomycin (BLM), an anti-tumor agent that can damage DNA by free radical mechanisms. Both chemicals have been previously shown to be mutagenic in the rat lymphocyte Hprt assay. Adult female Fischer 344 rats were given the antioxidants singly or in a combination 2 weeks prior to mutagen treatment. Antioxidant intake continued for an additional 4 weeks post-mutagen treatment. At sacrifice, spleens were aseptically removed for the isolation of lymphocytes to conduct the mutagenesis assay at the Hprt locus. The DMBA and BLM treatment induced a marked increase in MF, 52.8 x 10(-6) and 19.2 x 10(-6), respectively, over the controls. The MFs seen in the individual antioxidants alone (single or mixture) were relatively similar to the controls, with the exception of Vitamins C and E, that had 1.7- and 1.5-fold increase, respectively. The degree of inhibitory response was dependent on the type of mutagen and the particular antioxidant. BLM/antioxidant combination had inhibitions ranging from 44 to 80%, while DMBA/antioxidant system ranged from 60 to 93%, with Vitamins C and E achieving the highest inhibition in both systems. The mixture displayed low inhibitory responses, 44.6% for BLM/mix and 47% DMBA/mix. On the whole, the results indicate that the dietary constituents tested are antimutagenic; however, because of the gradations seen with the responses, the protective efficacy of these antioxidants may depend on the type of mutagen/carcinogen they encounter. Pending molecular analysis of mitochondrial DNA mutations will also indicate whether there is a shift in the mutational spectra produced by the carcinogens in the presence of antioxidants.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1996
Interactions between cloning efficiency (CE) and mutant frequency (MF) in the HPRT clonal assay i... more Interactions between cloning efficiency (CE) and mutant frequency (MF) in the HPRT clonal assay in in vitro study were analysed. In 12 separate reconstruction experiments with independent pairs of wild type (WT) and mutant (HPRT-) clones, the CE of WT cells (Group 1) and the recovery of mutant cells in absence (Group 2), as well as in the presence of non-irradiated (Group 3), or irradiated (Group 4) WT cells (IO' cells/well) was determined. The plating of mutant cells with irradiated WT cells improved their CEs by almost 30%. In contrast, the presence of non-irradiated WT cells led to a slight decline (10%) in CE of mutant cells, resulting in a significant difference between groups (p = 0.0083). The extent of decline in survival of mutant cells in the presence of non-irradiated WT cells negatively correlated (r = 0.3496, p < 0.05) with the initial CE of WT cells. The data suggest that the presence of WT cells in the selection plates may suppress the recovery of mutants in HPRT assay, and this negative effect is stronger in samples with high CE. These findings indicate a possible source for a serious underestimation of mutant frequencies (3-fold in the range of CEs from 10% to 60%) in the HPRT assay and may be useful for the interpretation of results from studies on exposure to mutagens in humans.
Circulation, Nov 22, 2011
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2002
In our previous studies, we have shown the mutagenicity of bleomycin (BLM) at the nuclear hprt lo... more In our previous studies, we have shown the mutagenicity of bleomycin (BLM) at the nuclear hprt locus. In the present study we have analyzed mutagenic effects of BLM in mitochondrial DNA (mtDNA) using short extension-PCR (SE-PCR) method for detection of low-copy deletions. Fisher 344 rats were treated with a single dose of BLM and total DNA preparations from splenic lymphocytes were processed in SE-PCR assay. Spontaneous deletions were typically flanked by direct repeats (78.5%), while the in BLM-treated group, direct repeats were found in only 46.6% of breakpoints. The ratio between deletions based on direct repeats and random sequence deletions changed from 3.67 in control group to 0.87 in BLM-treated animals, which corresponds to an approximate 1.7-fold increase in the deletion mutation frequency. Furthermore, 62.5% of deletions not flanked by direct repeats in the treated group contained cleavage sites for BLM. The localization of breakpoints was not entirely random. We have found four clusters containing deletions from both groups indicative of deletion hot spots. The results indicate that BLM exposure may be associated with the induction of mtDNA mutations, and suggest the utility of SE-PCR method for evaluating drug-induced genotoxicity.
Molecular neurobiology, 2015
Toll-like receptors (TLRs) play an essential role in innate immune response. Expression of TLRs h... more Toll-like receptors (TLRs) play an essential role in innate immune response. Expression of TLRs has also been linked to autophagy. As the main receptor for oxidized low-density lipoprotein (ox-LDL) on the cell surface, lectin-like ox-LDL receptor-1 (LOX-1) is upregulated by proinflammatory cytokines and has been linked to the development of autophagy. However, the relationship between LOX-1, autophagy, and TLR4 in neurons has not been defined. Here, we show that Angiotensin II (Ang II) treatment of CATH.a differentiated neuronal cells resulted in the expression of TLR4 (and associated signals MyD88 and Toll/interleukin-1 receptor domain-containing adapter-inducing interferon (TRIF)), LOX-1 autophagy. LOX-1 knockdown (transfection with specific small interfering RNA (siRNA)) resulted in reduced expression of TLR4 (and associated signals MyD88 and TRIF) and P-P38 mitogen-activated protein kinase (MAPK) and autophagy. TLR4 knockdown with siRNA resulted in reduced LOX-1 expression and a...
Cardiovascular Drugs and Therapy, 2014
Lectin-like oxidized low-density lipoprotein (ox-LDL) receptor-1 (LOX-1) is a major receptor for ... more Lectin-like oxidized low-density lipoprotein (ox-LDL) receptor-1 (LOX-1) is a major receptor for ox-LDL in endothelial cells. Its activation regulates endothelial proliferation, differentiation, migration and apoptosis. Recent in vitro studies show that LOX-1 activation by ox-LDL and angiotensin II (Ang II) induces angiogenesis via activation of NADPH oxidase and subsequent increase in ROS production. In this study, we investigated the effect of LOX-1 gene deletion (LOX-1 knockout or KO mice) on angiogenesis in response to prolonged Ang II infusion in vivo. Our studies showed that Ang II (vs. saline) infusion enhanced capillary formation in subcutaneously injected Matrigel® plugs. Ang II infusion also resulted in marked angiogenesis in the hearts as determined by CD31 immunopositivity. There was an increased expression (RT-PCR and Western blotting) of CD31 and VEGF in the hearts of mice infused with Ang II, indicating pro-angiogenic miliue. More importantly, LOX-1 KO mice reveled markedly limited angiogenesis in the Matrigel® plugs as well as in the hearts despite similar infusion with Ang II (all P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05 vs. wild-type mice). In addition, the hearts of LOX-1 KO mice had attenuated expression of pro-inflammatory and angiogenic signals MCP-1 and IL-1β following Ang II Infusion. Lastly, the rise in blood pressure in response to Ang II was less in the LOX-1 KO mice (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05 vs. wild-type mice). Our findings suggest that LOX-1 participates in angiogenesis in hypertension, which may be related to a state of inflammation.
American journal of physiology. Heart and circulatory physiology, Jan 15, 2014
Myocardial infarct size is determined by the death of nonischemic border zone cardiomyocytes caus... more Myocardial infarct size is determined by the death of nonischemic border zone cardiomyocytes caused by export of injury signals from the infarct zone. The countermeasures to limit infarct size, therefore, should be aimed at nonselective blockade of most, if not all, injury signals from entering nonischemic cells. To test whether inhibition of endocytosis might limit infarct size, HL-1 cardiomyocytes were subjected to anoxia (6 h) and reoxygenation (1 h). Anoxic and reoxygenated cells showed a multifold increase in mitochondrial ROS production accompanied with upregulation of scavenger receptors lectin-like oxidized low-density lipoprotein receptor-1 and CD36 and stimulation of stress signals, including NADPH oxidase subunit p22(phox), SOD2, and beclin-1. Incubation of healthy cardiomyocytes in media from anoxic and reoxygenated cells (conditioned media) resulted in qualitatively similar responses, including increase in the generation of mitochondrial ROS, p22(phox), SOD2, and beclin...
Journal of Molecular and Cellular Cardiology, 2015
Endothelin-1 (ET-1) plays a major role in regulating myocardial fibrosis in several pathological ... more Endothelin-1 (ET-1) plays a major role in regulating myocardial fibrosis in several pathological conditions, such as hypertension and diabetes. Aging is an independent risk factor for myocardial fibrosis. We hypothesized that ET-1 upregulation may be a basis of enhanced collagen synthesis in the senescent fibroblasts resulting in cardiac fibrosis with aging. To examine this hypothesis, we cultured mouse cardiac fibroblasts to passage-30 (P30). β-Galactosidase activity and several other aging markers were markedly increased in P30 (vs. P3) fibroblasts, indicating that these cells were indeed undergoing senescence. Importantly, ET-1 expression was markedly upregulated in P30 (vs. P3) fibroblasts. Of note, estrogen receptor-α (ER-α), an important negative regulator of ET-1, was downregulated in P30 fibroblasts. We also studied aged (130-weeks old, female) mice hearts, and observed that ET-1 was upregulated and ER-α was downregulated in these hearts (vs. 6-week old mice hearts, female). Similar observations were made in the fibroblasts isolated from aged mice hearts. ET-1 upregulation with aging was also seen in ≈70-year old (vs. ≈30-year old) human heart sections. In concert with ET-1 upregulation, the expression of fibronectin and collagens was found to be markedly increased in P30 cardiac fibroblasts in culture, fibroblasts isolated from the aged mice hearts, and in aged human hearts. Interestingly, inhibition of ET-1 in the senescent P30 fibroblasts by 2 different strategies (the use of siRNA and the use of endothelin converting enzyme inhibitors) markedly suppressed expression of fibrosis signals. Further, treatment with synthetic ET-1 enhanced fibronectin and collagen expression in P3 cardiac fibroblasts. These observations in mice and human hearts suggest that aging-related cardiac fibrosis is, at least partially, dependent on the upregulation of ET-1.
Human Mutation, 1997
The relative contribution of both genetic and environmental factors to spontaneous mutation frequ... more The relative contribution of both genetic and environmental factors to spontaneous mutation frequency in humans is unknown. We have investigated the contribution of genetic factors to this phenomenon by determining the in vivo mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in circulating T-lymphocytes obtained from pairs of monozygotic twins. hprt mutant frequencies were determined three times over fourteen days in six sets of monozygotic male twins (mean age 30) taking part in a Russian Space Program inclined bed rest experiment. Blood samples were obtained prior to, during, and immediately following the experiment. Mononuclear cells were separated, frozen, and flown to Canada for analysis using the hprt T-lymphocyte clonal assay. There is no evidence within this data set to demonstrate that the period of inclined bed rest to simulate the effects of weightlessness had any effect on the observed mutant frequency. However, the average mutant frequency for the six sets of Russian twins was found to be three times higher than that of Western counterparts. More surprisingly, the spontaneous mutant frequency of monozygotic twins was found to be much more similar within pairs than between pairs of twins. These data suggest that the contribution of genetics in the determination of mutation frequency is substantial. However, whether high concordance within twin pairs reflects shared environmental experience as well as common genetic factors is not entirely clear. More data will be required to distinguish genetic from environmental factors and to determine the degree to which mutant frequency is genetically determined.
Mechanisms of Ageing and Development, 2006
Mitochondria have long been suspected to be among the leading determinants of aging due to their ... more Mitochondria have long been suspected to be among the leading determinants of aging due to their functional importance and accelerated deterioration caused by accumulation of mutations in the mitochondrial DNA. Direct repeats are known to contribute to deletion formation in mtDNA and are a powerful source of reactive oxygen species (ROS)-independent mutagenesis. To evaluate the potential importance of homology-based deletion formation, we have analyzed the association between direct repeats in the mtDNA sequence and the lifespans of 65 mammalian species. Here, we report a significant negative correlation between the mutagenic potential of direct repeats and the mammalian lifespan, which is especially evident in closely related species.
Journal of the American College of Cardiology, 2011
Background: Activation of LOX-1, a lectin-like ox-LDL receptor-1, exerts a significant role in co... more Background: Activation of LOX-1, a lectin-like ox-LDL receptor-1, exerts a significant role in collagen formation. We hypothesized that LOX-1 deletion may inhibit inflammatory/oxidative stress signals and reduce collagen accumulation, and attenuate cardiac remodeling after chronic ischemia.
Experimental Gerontology, 2005
This study analyzed the incidence of point mutations in mitochondrial DNA of brain and muscle tis... more This study analyzed the incidence of point mutations in mitochondrial DNA of brain and muscle tissues from young (6-month) and old (24-month) male F344 rats. Coding sequence mutations in subunit 5 of the NADH dehydrogenase gene were detected after high-fidelity PCR amplification and cloning by denaturing gradient gel electrophoresis (DGGE) assay followed by sequencing of detected mutants. In total, almost a thousand individual clones were analyzed both in brain and muscle samples. On average, mtDNA from brain tissue showed a 66% increase with age in mutation frequencies (2.3G1.9 vs. 3.8G4.5!10 K4 mutations/bp, meanGSD), which failed to reach statistical significance (pZ0.45). Muscle tissues yielded substantially fewer mutants with average mutant frequencies for both young and old rats almost 10 times lower than the corresponding values in the brain tissue (0.3G0.4 and 0.5G0.6!10 K4 , respectively). The difference in mutation accumulation between muscle and brain was highly significant in both the younger group (Chi-squaredZ9.7, p%0.01) and in older animals (Chi-squaredZ10.9, p%0.001). Molecular analysis of the mutated sequences revealed that almost half were identical sequences recurring in different samples and tissues. Our findings indicate that the process of mutation accumulation in mitochondria begins in the germ-line and/or during earlier stages of life, contributing up to half of the total mutational burden, whereas de novo spontaneous formation of point mutations in adulthood is far less than was anticipated. q 2005 Elsevier Inc. All rights reserved.
Accumulating evidence suggests that the aging process is, in part, driven by accumulation of larg... more Accumulating evidence suggests that the aging process is, in part, driven by accumulation of large deletions in mitochondrial DNA (mtDNA). Here, I present a hypothesis that significant variations in lifespans can be explained by species-specific mtDNA sequence features that cause a shift in the mode of mtDNA replication and thus preclude the formation of large deletions.
PLoS ONE, 2012
Dyslipidemia and obesity are primary risk factors for the development of atherosclerosis and are ... more Dyslipidemia and obesity are primary risk factors for the development of atherosclerosis and are also epidemiologically linked to increased susceptibility to a variety of cancers including breast cancer. One of the prominent features of dyslipidemia is enhanced production of oxidized LDL (ox-LDL), which has been shown to be implicated in key steps of atherogenesis including inflammatory signaling and proliferation of vascular cells. In this study we analyzed the effects of ox-LDL in human mammary epithelial cells (MCF10A). MCF10A cells avidly internalized dil-ox-LDL and exhibited increased proliferative response to ox-LDL within the range of 1-50 mg/ml in a dose-dependent manner. Treatment of cells with 20 mg/ml ox-LDL for 2 and 12 hours was associated with upregulation of LOX-1 and CD36 scavenger receptors while MSR1 and CXLC16 receptors did not change. Ox-LDL-treated cells displayed significant upregulation of NADPH oxidases (subunits P22 phox and P47 phox ), lipoxygenases-12 and -15, and cytoplasmic, but not mitochondrial, SOD. Ox-LDL also triggered phosphorylation of IkBa coupled with nuclear translocation of NF-kB and stimulated p44/42 MAPK, PI3K and Akt while intracellular PTEN (PI3K/Akt pathway inhibitor and target of miR-21) declined. Quantitative PCR revealed increased expression of hsa-miR-21 in ox-LDL treated cells coupled with inhibition of miR-21 target genes. Further, transfection of MCF10A cells with miR-21 inhibitor prevented ox-LDL mediated stimulation of PI3K and Akt. We conclude that, similarly to vascular cells, mammary epithelial cells respond to ox-LDL by upregulation of proliferative and pro-inflammatory signaling. We also report for the first time that part of ox-LDL triggered reactions in MCF10A cells is mediated by oncogenic hsa-miR-21 through inhibition of its target gene PTEN and consequent activation of PI3K/Akt pathway.
Biochemical Basis and Therapeutic Implications of Angiogenesis, 2013
PLoS ONE, 2011
Numerous studies have described the process of senescence associated with accumulation of oxidati... more Numerous studies have described the process of senescence associated with accumulation of oxidative damage, mutations and decline in proliferative potential. Although the changes observed in senescent cells are likely to result in significant phenotypic alterations, the studies on consequences of endothelial senescence, especially in relation to aging-associated diseases, are scarce. We have analyzed effects of senescence on the functions of endothelial cells relevant to the development of atherosclerosis including angiogenesis, adhesion, apoptosis and inflammation. In the course of progressing through the passages, human umbilical vein endothelial cells (HUVECs) displayed significant increase in size (+36% passage 12 vs. passage 4 , p,0.001) and reduction in both basal and VEGF-stimulated tube formation. The analysis of a scavenger receptor LOX-1, a key molecule implicated in atherogenesis, revealed a significant decline of its message (mRNA) and protein content in senescent endothelial cells (233%) and in aortas of 50 wk (vs. 5 wk) old mice (all p,0.01). These effects were accompanied by a marked reduction of the basal expression of VCAM-1 and ICAM-1. Compared to early cultures, late passage HUVECs also exhibited nuclear translocation of NF-kB (p65) and reciprocal shifts in BAX and BCL2 protein content resulting in almost 2-fold increase in BAX/BCL2 ratio and 3-fold increase in apoptotic response to TNFa exposure (p,0.04). These changes in senescent endothelial cells are suggestive of aberrant responses to physiological stimuli resulting in a less permissive environment for tissue remodeling and progression of diseases requiring angiogenesis and cell adhesion in elderly, possibly, mediated by LOX-1.
Cardiovascular Drugs and Therapy, 2011
... Oncogene. 2004;23:636578. 3. Lorincz AM, Sukumar S. Molecular links between obesity and brea... more ... Oncogene. 2004;23:636578. 3. Lorincz AM, Sukumar S. Molecular links between obesity and breast cancer. Endocr Relat Canc. 2006;13:27992. 4. Mehta JL. ... Physiol Pharma-col. 2000;51:88396. 15. Roberts CK, Barnard RJ, Sindhu RK, Jurczak M, Ehdaie A, Vaziri ND. ...
Cardiovascular research, 2014
Journal of cardiovascular pharmacology and therapeutics, 2011
Hypoxia-reoxygenation (HR) is a major driver for angiogenesis in atherosclerotic plaques and tumo... more Hypoxia-reoxygenation (HR) is a major driver for angiogenesis in atherosclerotic plaques and tumors. Angiogenesis is a multistep process requiring stimulation of proliferation and migration of endothelial cells in response to a number of growth factors, including transforming growth factor (TGFβ1). Aspirin (acetylsalicylic acid) has been shown to reduce atherosclerosis-related events as well as development of certain tumors. We examined the role of aspirin in HR-mediated angiogenesis from human umbilical vein endothelial cells (HUVECs). We found that aspirin (0.5 and 1 mmol/L) markedly (by about 30%, P < .01) reduced HR-mediated tube formation. Next, we studied changes in TGFβ1 and its type 1 receptor (TGFβ-R1) as well as in the transcription of p53 and p21 during HR-mediated angiogenesis. Hypoxia-reoxygenation modestly increased TGFβ1 and decreased its type 1 receptor (TGFβ-R1) transcription (both P-NS) and reduced the transcription of p53 and p21 (P < .05). Treatment of HUVE...
Molecular cancer, 2005
Several studies of tumors have revealed substantial numbers of clonally expanded somatic mutation... more Several studies of tumors have revealed substantial numbers of clonally expanded somatic mutations in mitochondrial DNA (mtDNA), not observed in adjacent intact tissues. These findings were interpreted as indicating the involvement of mtDNA mutations in tumorigenesis. Such comparisons, however, ignore an important confounding factor: the monoclonal origin of tumors as opposed to the highly polyclonal nature of normal tissues. Analysis of recently published data on the incidence of somatic mutations in nontumor monoclonal cells suggests that, contrary to the prevailing view, the process of tumorigenesis may be accompanied by active selection against detrimental mtDNA mutations.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2001
Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals prov... more Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals provide end points that can be used for assessing the antimutagenic or anticarcinogenic properties of putative chemopreventive compounds and for predicting their protective efficacy in humans. In this study, we investigated the ability of the dietary antioxidant Vitamins C, E, beta-carotene and the mineral selenium to inhibit the mutant frequency (MF) induced by treatment of rats with 7,12-dimethylbenz[a]anthracene (DMBA), a mammary carcinogen and bleomycin (BLM), an anti-tumor agent that can damage DNA by free radical mechanisms. Both chemicals have been previously shown to be mutagenic in the rat lymphocyte Hprt assay. Adult female Fischer 344 rats were given the antioxidants singly or in a combination 2 weeks prior to mutagen treatment. Antioxidant intake continued for an additional 4 weeks post-mutagen treatment. At sacrifice, spleens were aseptically removed for the isolation of lymphocytes to conduct the mutagenesis assay at the Hprt locus. The DMBA and BLM treatment induced a marked increase in MF, 52.8 x 10(-6) and 19.2 x 10(-6), respectively, over the controls. The MFs seen in the individual antioxidants alone (single or mixture) were relatively similar to the controls, with the exception of Vitamins C and E, that had 1.7- and 1.5-fold increase, respectively. The degree of inhibitory response was dependent on the type of mutagen and the particular antioxidant. BLM/antioxidant combination had inhibitions ranging from 44 to 80%, while DMBA/antioxidant system ranged from 60 to 93%, with Vitamins C and E achieving the highest inhibition in both systems. The mixture displayed low inhibitory responses, 44.6% for BLM/mix and 47% DMBA/mix. On the whole, the results indicate that the dietary constituents tested are antimutagenic; however, because of the gradations seen with the responses, the protective efficacy of these antioxidants may depend on the type of mutagen/carcinogen they encounter. Pending molecular analysis of mitochondrial DNA mutations will also indicate whether there is a shift in the mutational spectra produced by the carcinogens in the presence of antioxidants.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1996
Interactions between cloning efficiency (CE) and mutant frequency (MF) in the HPRT clonal assay i... more Interactions between cloning efficiency (CE) and mutant frequency (MF) in the HPRT clonal assay in in vitro study were analysed. In 12 separate reconstruction experiments with independent pairs of wild type (WT) and mutant (HPRT-) clones, the CE of WT cells (Group 1) and the recovery of mutant cells in absence (Group 2), as well as in the presence of non-irradiated (Group 3), or irradiated (Group 4) WT cells (IO' cells/well) was determined. The plating of mutant cells with irradiated WT cells improved their CEs by almost 30%. In contrast, the presence of non-irradiated WT cells led to a slight decline (10%) in CE of mutant cells, resulting in a significant difference between groups (p = 0.0083). The extent of decline in survival of mutant cells in the presence of non-irradiated WT cells negatively correlated (r = 0.3496, p < 0.05) with the initial CE of WT cells. The data suggest that the presence of WT cells in the selection plates may suppress the recovery of mutants in HPRT assay, and this negative effect is stronger in samples with high CE. These findings indicate a possible source for a serious underestimation of mutant frequencies (3-fold in the range of CEs from 10% to 60%) in the HPRT assay and may be useful for the interpretation of results from studies on exposure to mutagens in humans.
Circulation, Nov 22, 2011
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2002
In our previous studies, we have shown the mutagenicity of bleomycin (BLM) at the nuclear hprt lo... more In our previous studies, we have shown the mutagenicity of bleomycin (BLM) at the nuclear hprt locus. In the present study we have analyzed mutagenic effects of BLM in mitochondrial DNA (mtDNA) using short extension-PCR (SE-PCR) method for detection of low-copy deletions. Fisher 344 rats were treated with a single dose of BLM and total DNA preparations from splenic lymphocytes were processed in SE-PCR assay. Spontaneous deletions were typically flanked by direct repeats (78.5%), while the in BLM-treated group, direct repeats were found in only 46.6% of breakpoints. The ratio between deletions based on direct repeats and random sequence deletions changed from 3.67 in control group to 0.87 in BLM-treated animals, which corresponds to an approximate 1.7-fold increase in the deletion mutation frequency. Furthermore, 62.5% of deletions not flanked by direct repeats in the treated group contained cleavage sites for BLM. The localization of breakpoints was not entirely random. We have found four clusters containing deletions from both groups indicative of deletion hot spots. The results indicate that BLM exposure may be associated with the induction of mtDNA mutations, and suggest the utility of SE-PCR method for evaluating drug-induced genotoxicity.
Molecular neurobiology, 2015
Toll-like receptors (TLRs) play an essential role in innate immune response. Expression of TLRs h... more Toll-like receptors (TLRs) play an essential role in innate immune response. Expression of TLRs has also been linked to autophagy. As the main receptor for oxidized low-density lipoprotein (ox-LDL) on the cell surface, lectin-like ox-LDL receptor-1 (LOX-1) is upregulated by proinflammatory cytokines and has been linked to the development of autophagy. However, the relationship between LOX-1, autophagy, and TLR4 in neurons has not been defined. Here, we show that Angiotensin II (Ang II) treatment of CATH.a differentiated neuronal cells resulted in the expression of TLR4 (and associated signals MyD88 and Toll/interleukin-1 receptor domain-containing adapter-inducing interferon (TRIF)), LOX-1 autophagy. LOX-1 knockdown (transfection with specific small interfering RNA (siRNA)) resulted in reduced expression of TLR4 (and associated signals MyD88 and TRIF) and P-P38 mitogen-activated protein kinase (MAPK) and autophagy. TLR4 knockdown with siRNA resulted in reduced LOX-1 expression and a...
Cardiovascular Drugs and Therapy, 2014
Lectin-like oxidized low-density lipoprotein (ox-LDL) receptor-1 (LOX-1) is a major receptor for ... more Lectin-like oxidized low-density lipoprotein (ox-LDL) receptor-1 (LOX-1) is a major receptor for ox-LDL in endothelial cells. Its activation regulates endothelial proliferation, differentiation, migration and apoptosis. Recent in vitro studies show that LOX-1 activation by ox-LDL and angiotensin II (Ang II) induces angiogenesis via activation of NADPH oxidase and subsequent increase in ROS production. In this study, we investigated the effect of LOX-1 gene deletion (LOX-1 knockout or KO mice) on angiogenesis in response to prolonged Ang II infusion in vivo. Our studies showed that Ang II (vs. saline) infusion enhanced capillary formation in subcutaneously injected Matrigel® plugs. Ang II infusion also resulted in marked angiogenesis in the hearts as determined by CD31 immunopositivity. There was an increased expression (RT-PCR and Western blotting) of CD31 and VEGF in the hearts of mice infused with Ang II, indicating pro-angiogenic miliue. More importantly, LOX-1 KO mice reveled markedly limited angiogenesis in the Matrigel® plugs as well as in the hearts despite similar infusion with Ang II (all P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05 vs. wild-type mice). In addition, the hearts of LOX-1 KO mice had attenuated expression of pro-inflammatory and angiogenic signals MCP-1 and IL-1β following Ang II Infusion. Lastly, the rise in blood pressure in response to Ang II was less in the LOX-1 KO mice (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05 vs. wild-type mice). Our findings suggest that LOX-1 participates in angiogenesis in hypertension, which may be related to a state of inflammation.
American journal of physiology. Heart and circulatory physiology, Jan 15, 2014
Myocardial infarct size is determined by the death of nonischemic border zone cardiomyocytes caus... more Myocardial infarct size is determined by the death of nonischemic border zone cardiomyocytes caused by export of injury signals from the infarct zone. The countermeasures to limit infarct size, therefore, should be aimed at nonselective blockade of most, if not all, injury signals from entering nonischemic cells. To test whether inhibition of endocytosis might limit infarct size, HL-1 cardiomyocytes were subjected to anoxia (6 h) and reoxygenation (1 h). Anoxic and reoxygenated cells showed a multifold increase in mitochondrial ROS production accompanied with upregulation of scavenger receptors lectin-like oxidized low-density lipoprotein receptor-1 and CD36 and stimulation of stress signals, including NADPH oxidase subunit p22(phox), SOD2, and beclin-1. Incubation of healthy cardiomyocytes in media from anoxic and reoxygenated cells (conditioned media) resulted in qualitatively similar responses, including increase in the generation of mitochondrial ROS, p22(phox), SOD2, and beclin...
Journal of Molecular and Cellular Cardiology, 2015
Endothelin-1 (ET-1) plays a major role in regulating myocardial fibrosis in several pathological ... more Endothelin-1 (ET-1) plays a major role in regulating myocardial fibrosis in several pathological conditions, such as hypertension and diabetes. Aging is an independent risk factor for myocardial fibrosis. We hypothesized that ET-1 upregulation may be a basis of enhanced collagen synthesis in the senescent fibroblasts resulting in cardiac fibrosis with aging. To examine this hypothesis, we cultured mouse cardiac fibroblasts to passage-30 (P30). β-Galactosidase activity and several other aging markers were markedly increased in P30 (vs. P3) fibroblasts, indicating that these cells were indeed undergoing senescence. Importantly, ET-1 expression was markedly upregulated in P30 (vs. P3) fibroblasts. Of note, estrogen receptor-α (ER-α), an important negative regulator of ET-1, was downregulated in P30 fibroblasts. We also studied aged (130-weeks old, female) mice hearts, and observed that ET-1 was upregulated and ER-α was downregulated in these hearts (vs. 6-week old mice hearts, female). Similar observations were made in the fibroblasts isolated from aged mice hearts. ET-1 upregulation with aging was also seen in ≈70-year old (vs. ≈30-year old) human heart sections. In concert with ET-1 upregulation, the expression of fibronectin and collagens was found to be markedly increased in P30 cardiac fibroblasts in culture, fibroblasts isolated from the aged mice hearts, and in aged human hearts. Interestingly, inhibition of ET-1 in the senescent P30 fibroblasts by 2 different strategies (the use of siRNA and the use of endothelin converting enzyme inhibitors) markedly suppressed expression of fibrosis signals. Further, treatment with synthetic ET-1 enhanced fibronectin and collagen expression in P3 cardiac fibroblasts. These observations in mice and human hearts suggest that aging-related cardiac fibrosis is, at least partially, dependent on the upregulation of ET-1.
Two or three times a year, principal investigators closet themselves in their offices for about a... more Two or three times a year, principal investigators closet themselves in their offices for about a month to prepare grant applications for National Institutes of Health (NIH) or similar agencies to ensure funding. It is no longer research as usual but rather a feverish production of pictures, graphs and preliminary experimental data to be incorporated into proposals to make them more attractive. It is a matter of life and death because, somehow, NIH awards have become not only the icing on the cake as was originally intended, but the entire cake. The National Institutes of Health had a rather humble beginning as a bacteriological laboratory "for investigating the origin and causes of epidemic diseases, especially yellow fever and cholera" in 1878. As is typical for all pre-cancerous lesions, the early stages of NIH evolution were rather benign until the creation of the Research Grants Office within NIH in 1946. This office was charged with administering a program of extramural research grants and fellowship awards, with multiple study sections for review of research grant applications. Today, NIH is a monstrously big and complex organization that controls every facet of scientific activity (and, incidentally, livelihood of all scientists) in the United States through awarding or withholding funding for research, presumably, based on merit determined by peer review.
This is an already submitted petition to abolish NIH as a primary funding mechanism for biomedica... more This is an already submitted petition to abolish NIH as a primary funding mechanism for biomedical science. If you agree with my reasoning, please sign it.
Best regards to all,
M.Khaidakov, PhD