Publications by authors named "Asmat Salim"

32 Publications

Umbilical cord-derived mesenchymal stem cells preconditioned with isorhamnetin: potential therapy for burn wounds.

World J Stem Cells 2020 Dec;12(12):1652-1666

Stem Cell Laboratory, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 74700, Sindh, Pakistan.

Background: Impaired wound healing can be associated with different pathological states. Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide. Mesenchymal stem cells (MSCs) possess the ability to regenerate tissues by secreting factors involved in promoting cell migration, proliferation and differentiation, while suppressing immune reactions. Preconditioning of MSCs with small molecules having cytoprotective properties can enhance the potential of these cells for their use in cell-based therapeutics.

Aim: To enhance the therapeutic potential of MSCs by preconditioning them with isorhamnetin for second degree burn wounds in rats.

Methods: Human umbilical cord MSCs (hU-MSCs) were isolated and characterized by surface markers, CD105, vimentin and CD90. For preconditioning, hU-MSCs were treated with isorhamnetin after selection of the optimized concentration (5 µmol/L) by cytotoxicity analysis. The migration potential of these MSCs was analyzed by the scratch assay. The healing potential of normal, and preconditioned hU-MSCs was compared by transplanting these MSCs in a rat model of a second degree burn wound. Normal, and preconditioned MSCs (IH + MSCs) were transplanted after 72 h of burn injury and observed for 2 wk. Histological and gene expression analyses were performed on day 7 and 14 after cell transplantation to determine complete wound healing.

Results: The scratch assay analysis showed a significant reduction in the scratch area in the case of IH + MSCs compared to the normal untreated MSCs at 24 h, while complete closure of the scratch area was observed at 48 h. Histological analysis showed reduced inflammation, completely remodeled epidermis and dermis without scar formation and regeneration of hair follicles in the group that received IH + MSCs. Gene expression analysis was time dependent and more pronounced in the case of IH + MSCs. Interleukin (IL)-1β, IL-6 and Bcl-2 associated X genes showed significant downregulation, while transforming growth factor β, vascular endothelial growth factor, Bcl-2 and matrix metallopeptidase 9 showed significant upregulation compared to the burn wound, showing increased angiogenesis and reduced inflammation and apoptosis.

Conclusion: Preconditioning of hU-MSCs with isorhamnetin decreases wound progression by reducing inflammation, and improving tissue architecture and wound healing. The study outcome is expected to lead to an improved cell-based therapeutic approach for burn wounds.
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http://dx.doi.org/10.4252/wjsc.v12.i12.1652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789118PMC
December 2020

Effect of valproic acid on the hepatic differentiation of mesenchymal stem cells in 2D and 3D microenvironments.

Mol Cell Biochem 2021 Feb 27;476(2):909-919. Epub 2020 Oct 27.

Dr. Zafar H. Zaidi Center for Proteomics, University of Karachi, Karachi, 75270, Pakistan.

Mesenchymal stem cells (MSCs) have multi-lineage differentiation potential which make them an excellent source for cell-based therapies. Histone modification is one of the major epigenetic regulations that play central role in stem cell differentiation. Keeping in view their ability to maintain gene expression essential for successful differentiation, it was interesting to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, in the hepatic differentiation of MSCs within the 3D scaffold. MSCs were treated with the optimized concentration of VPA in the 3D collagen scaffold. Analyses of hepatic differentiation potential of treated MSCs were performed by qPCR, immunostaining and periodic acid Schiff assay. Our results demonstrate that MSCs differentiate into hepatic-like cells when treated with 5 mM VPA for 24 h. The VPA-treated MSCs have shown significant upregulation in the expression of hepatic genes, CK-18 (P < 0.05), TAT (P < 0.01), and AFP (P < 0.001), and hepatic proteins, AFP (P < 0.05) and ALB (P < 0.01). In addition, acetylation of histones (H3 and H4) was significantly increased (P < 0.001) in VPA-pretreated cells. Further analysis showed that VPA treatment significantly enhanced (P < 0.01) glycogen storage, an important functional aspect of hepatic cells. The present study revealed the effectiveness of VPA in hepatic differentiation within the 3D collagen scaffold. These hepatic-like cells may have an extended clinical applicability in future for successful liver regeneration.
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http://dx.doi.org/10.1007/s11010-020-03955-9DOI Listing
February 2021

Effect of a dianthin G analogue in the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes.

Mol Cell Biochem 2020 Dec 31;475(1-2):27-39. Epub 2020 Jul 31.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan.

Loss of cardiomyocytes due to myocardial infarction results in ventricular remodeling which includes non-contractile scar formation, which can lead to heart failure. Stem cell therapy aims to replace the scar tissue with the functional myocardium. Mesenchymal stem cells (MSCs) are undifferentiated cells capable of self-renewal as well as differentiation into multiple lineages. MSCs can be differentiated into cardiomyocytes by treating them with small molecules and peptides. Here, we report for the first time, the role of a cyclic peptide, an analogue of dianthin G, [Glu]-dianthin G (1) in the in vitro cardiac differentiation of rat bone marrow MSCs. In this study, [Glu]-dianthin G (1) was synthesized using solid-phase total synthesis and characterized by NMR spectroscopy. MSCs were treated with two different concentrations (0.025 and 0.05 mM) of the peptide separately for 72 h and then incubated for 15 days to allow the cells to differentiate into cardiomyocytes. Treated cells were analyzed for the expression of cardiac-specific genes and proteins. Results showed significant upregulation of cardiac-specific genes GATA4, cardiac troponin T (cTnT), cardiac troponin I (cTnI), cardiac myosin heavy chain, and connexin 43 in the treated MSCs compared to the untreated control. For cardiac-specific proteins, GATA4, cTnT, and Nkx2.5 were analyzed in the treated cells and were shown to have significant upregulation as compared to the untreated control. In conclusion, this study has demonstrated the cardiac differentiation potential of [Glu]-dianthin G (1)-treated rat bone marrow MSCs in vitro both at the gene and at the protein levels. Transplantation of pre-differentiated MSCs into the infarcted myocardium may result in the efficient regeneration of cardiac cells and restoration of normal cardiac function.
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http://dx.doi.org/10.1007/s11010-020-03855-yDOI Listing
December 2020

Small molecule 2'-deoxycytidine differentiates human umbilical cord-derived MSCs into cardiac progenitors in vitro and their in vivo xeno-transplantation improves cardiac function.

Mol Cell Biochem 2020 Jul 15;470(1-2):99-113. Epub 2020 May 15.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan.

Small molecules are widely used to induce stem cell differentiation. 2'-deoxycytidine (2-DC) belongs to the cytidine family. It stimulates the expression of cardiac-specific genes and proteins, and directs mesenchymal stem cells towards cardiomyogenic differentiation. We aim to investigate the role of 2-DC-treated human umbilical cord mesenchymal stem cells (UC-MSCs) into myogenic lineage and explore their application in regeneration of infarcted myocardium. UC-MSCs were treated with 5, 10, 20, and 40 µM 2-DC following optimization by cytotoxicity analysis. Rat model of myocardial infarction (MI) was induced by ligating left anterior descending coronary artery. Normal, and 2-DC treated UC-MSCs were transplanted in the left ventricular wall immediately after ligation. Echocardiographic measurements were performed to assess cardiac function. Tissue architecture of the myocardium was examined by histological analysis to determine fate of the transplanted cells. MSCs were successfully isolated from human umbilical cord tissue. 2-DC treatment did not produce any significant cytotoxic effect in UC-MSCs at all concentrations. qPCR analysis of treated UC-MSCs showed induction of myogenic differentiation, which is more pronounced at 20 μM concentration. Fluorescently labeled 2-DC-treated UC-MSCs showed significant (**P < 0.01) homing in the infarcted myocardium as compared to normal UC-MSCs. Hearts transplanted with 2-DC-treated UC-MSCs significantly (***P < 0.001) improved the cardiac systolic and diastolic functions and pumping ability as compared to normal UC-MSCs and MI groups. Fibrotic area and left ventricular wall thickness were significantly improved (***P < 0.001) in 2-DC-treated group as compared to normal UC-MSCs. Immunohistochemical staining showed co-localization of fluorescently labeled cells and patches of differentiated myocytes which were stained for cardiac proteins in the infarct zone implying that the treated UC-MSCs regenerated cardiomyocytes. We report for the first time that 2-DC induces cardiac differentiation in UC-MSCs. Transplanted cells differentiated into functional cardiomyocytes and significantly improved cardiac performance. These pre-differentiated cardiac progenitors showed better survival, homing, and distribution in the infarcted zone. 2-DC treated cells not only improved cardiac function, but also restored tissue homeostasis, suggesting a better therapeutic option for the regeneration of cardiac tissue in the clinical setup.
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http://dx.doi.org/10.1007/s11010-020-03750-6DOI Listing
July 2020

IL-7 overexpression enhances therapeutic potential of rat bone marrow mesenchymal stem cells for diabetic wounds.

Wound Repair Regen 2019 05 6;27(3):235-248. Epub 2019 Mar 6.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan.

This study was aimed to enhance the healing potential of rat bone marrow mesenchymal stem cells against chronic diabetic wounds through interleukin-7 (IL-7) transfection. IL-7 plays an important role in wound healing and acts as a survival factor in some cell types. This study involves isolation, propagation, and characterization of mesenchymal stem cells (MSCs) and their modification with IL-7 gene via retroviral transfection. Transfected MSCs were assessed for their effect on angiogenic genes by qPCR. Wound healing potential of transfected MSCs was analyzed by scratch assay in vitro and by transplanting these cells in rat diabetic wound models in vivo. Wound area was measured for a period of 15 days and subsequent histological analysis was performed. qPCR results showed increased expression of IL-7 gene (p ≤ 0.05) and also principal angiogenic genes, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), VEGF receptor 1 (FLT-1), and VEGF receptor 2 (FLK-1) (p ≤ 0.05). Neuropilin-1 (NRP-1) did not show any significant change. In vitro analysis of IL-7 MSCs showed intense cell-cell connections and tube formation as compared to the normal MSCs. Rate of wound closure was more (p ≤ 0.001) in case of diabetic group transplanted with IL-7 MSCs. Histological examination revealed enhanced vascular supply in skin tissues of diabetic animals transplanted with IL-7 transfected MSCs as compared to normal MSCs. Immunohistochemical results showed significantly higher expression of IL-7 (p ≤ 0.001) and α-smooth muscle actin(p ≤ 0.001) in the tissue sections of IL-7 transfected group as compared to normal MSCs and the diabetic control group; the latter indicates increase in the number of blood vessels. It is concluded from this study that IL-7 overexpression in MSCs can enhance the healing potential of MSCs and aid in wound closure in diabetic animals through the induction of angiogenic genes.
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http://dx.doi.org/10.1111/wrr.12706DOI Listing
May 2019

Role of interleukin-7 in fusion of rat bone marrow mesenchymal stem cells with cardiomyocytes in vitro and improvement of cardiac function in vivo.

Cardiovasc Ther 2018 Dec 6;36(6):e12479. Epub 2018 Dec 6.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan.

Aims: Mesenchymal stem cells (MSCs) hold significant promise as potential therapeutic candidates following cardiac injury. However, to ensure survival of transplanted cells in ischemic environment, it is beneficial to precondition them with growth factors that play important role in cell survival and proliferation. Aim of this study is to use interleukin-7 (IL-7), a cell survival growth factor, to enhance the potential of rat bone marrow MSCs in terms of cell fusion in vitro and cardiac function in vivo.

Methods: Mesenchymal stem cells were transfected with IL-7 gene through retroviral vector. Normal and transfected MSCs were co-cultured with neonatal cardiomyocytes (CMs) and cell fusion was analyzed by flow cytometry and fluorescence microscopy. These MSCs were also transplanted in rat model of myocardial infarction (MI) and changes at tissue level and cardiac function were assessed by histological analysis and echocardiography, respectively.

Results: Co-culture of IL-7 transfected MSCs and CMs showed significantly higher (P < 0.01) number of fused cells as compared to normal MSCs. Histological analysis of hearts transplanted with IL-7 transfected MSCs showed significant reduction (P < 0.001) in infarct size and better preservation (P < 0.001) of left ventricular wall thickness as compared to normal MSCs. Presence of cardiac-specific proteins, α-actinin, and troponin-T showed that the transplanted MSCs were differentiated into cardiomyocytes. Echocardiographic recordings of the experimental group transplanted with transfected MSCs showed significant increase in the ejection fraction and fractional shortening (P < 0.01), and decrease in diastolic and systolic left ventricular internal diameters (P < 0.001) and end systolic and diastolic volumes (P < 0.01 and P < 0.001, respectively).

Conclusion: Interleukin-7 is able to enhance the fusogenic properties of MSCs and improve cardiac function. This improvement may be attributed to the supportive action of IL-7 on cell proliferation and cell survival contributing to the regeneration of damaged myocardium.
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http://dx.doi.org/10.1111/1755-5922.12479DOI Listing
December 2018

Effect of 2,4-Dinitrophenol preconditioning on the expression levels of mesenchymal markers in neonatal cardiac progenitors.

Hellenic J Cardiol 2017 Jan - Feb;58(1):98-102. Epub 2017 Feb 2.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan. Electronic address:

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http://dx.doi.org/10.1016/j.hjc.2017.01.007DOI Listing
July 2018

Epac-Rap1-activated mesenchymal stem cells improve cardiac function in rat model of myocardial infarction.

Cardiovasc Ther 2017 Apr;35(2)

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan.

Introduction: Rap1, a member of Ras superfamily of small GTP-binding proteins, is involved in cardiovascular biology in numerous ways. It is an evolutionary conserved regulator of adhesion, polarity, differentiation and growth.

Aims: Our aim was to analyze Rap1-activated rat bone marrow mesenchymal stem cells (MSCs) for their potential role in adhesion and cardiac differentiation.

Methods: Myocardial infarction (MI) was produced in Sprague Dawley (SD) rats through occlusion of the left anterior descending coronary artery. MSCs were treated with 8-pCPT-2'-O-Me-cAMP (CPT) to activate Rap1. Normal (untreated) and CPT-treated MSCs were transplanted through intramyocardial injection in respective groups. Cardiac function was assessed by echocardiography at 2 and 4 weeks after cell transplantation. Histological analysis was performed to observe changes at tissue level.

Results: Homing of CPT-treated MSCs was significantly (***P<.001) higher as compared to normal MSCs in the infarcted hearts. This may be due to increase in the gene expression of some of the cell adhesion molecules as evident by qRT-PCR analysis. Significant (***P<.001) improvement in the restoration of heart function in terms of left ventricular diastolic and systolic internal diameters (LVIDd, LVIDs), % ejection fraction, % fraction shortening and end-systolic and end-diastolic volumes were observed in CPT-treated MSCs as compared to the MI model. Histological analyses showed significant (***P<.001) reduction in scar formation in the CPT-treated group. Differentiation of treated MSCs into functional cardiomyocytes was evident through immunohistochemical staining. LV wall thickness was also preserved significantly (***P<.001). Blood vessel formation was more pronounced in CPT-treated group although both cell therapy groups showed significant increase as compared to MI model.

Conclusion: Our findings showed that pharmacological activation of Epac-Rap1 improves cardiac function through better survival, adhesion and differentiation of transplanted cells. Transplantation of these MSCs in the infarct area restored functional myocardium.
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http://dx.doi.org/10.1111/1755-5922.12248DOI Listing
April 2017

Conditioned media trans-differentiate mature fibroblasts into pancreatic beta-like cells.

Life Sci 2016 Nov 1;164:52-59. Epub 2016 Sep 1.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. Electronic address:

Aim: The study was carried out to evaluate the role of preconditioning strategies on the trans-differentiation of mature fibroblasts (NIH3T3 cells) into insulin producing β-cells.

Methods: The NIH3T3 cells were treated with dexamethasone (5μM) and pancreatic extract (0.05 and 0.4mg/mL) separately or in combination. The treated cells were analyzed for the morphological changes, and expression of pancreatic genes and proteins by phase contrast microscopy, RT-PCR and flow cytometry/immunocytochemistry, respectively.

Results: Treatment of mature fibroblasts with different combinations of dexamethasone and pancreatic extract in the form of conditioned media resulted in comparable morphological changes and expression of certain pancreatic genes and proteins; however, their expression varied with each treatment. Most prominent effect was observed in case of combined treatment which resulted in significant increase (p<0.001) in gene expression levels of insulin, MafA, and Ngn3. Variable pattern was observed in insulin, MafA, Ngn3 and Sca1 expressions at the protein level.

Conclusion: It is concluded from this study that preconditioning of NIH3T3 cells with conditioned media containing different combinations of dexamethasone and pancreatic extract can induce trans-differentiation of these cells into pancreatic β-like cells. The conditioned media however, need to be optimized. The study may offer the possibility of improved regeneration of mature cell type that could serve as a future therapeutic option for diabetes.
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http://dx.doi.org/10.1016/j.lfs.2016.08.032DOI Listing
November 2016

Preconditioning of mesenchymal stem cells with 2,4-dinitrophenol improves cardiac function in infarcted rats.

Life Sci 2016 Oct 17;162:60-9. Epub 2016 Aug 17.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, 75270 Karachi, Pakistan. Electronic address:

Aims: The aim of this study is to determine if preconditioning of bone marrow derived mesenchymal stem cells (MSCs) with 2,4-dinitrophenol (DNP) improves survival of transplanted stem cells in a rat model of myocardial infarction (MI), and to asses if this strategy has measurable impact on cardiac function.

Main Methods: MSCs were preconditioned with DNP. In vitro cell adhesion assay and qRT-PCR were performed to analyze the expression of genes involved in cardiomyogenesis, cell adhesion and angiogenesis. MI was produced by occlusion of left anterior descending coronary artery. One million cells were transplanted by intramyocardial injection into the infarcted myocardium. Echocardiography was performed after two and four weeks of cellular transplantation. Hearts were harvested after four weeks and processed for histological analysis.

Key Findings: DNP treated MSCs adhered to the surface more (p<0.001) as compared to the normal MSCs. Gene expression levels were significantly upregulated in case of DNP treatment. The number of viable MSCs was more (p<0.001) in animals that received DNP treated MSCs, leading to significant improvement in cardiac function. Histological analysis revealed significant reduction in scar formation (p<0.001), maintenance of left ventricular wall thickness (p<0.001), and increased angiogenesis (p<0.01).

Significance: The study evidenced for the first time that MSCs preconditioned with DNP improved cardiac function after transplantation. This can be attributed to improved survival, homing, adhesion, and cardiomyogenic and angiogenic differentiation of DNP treated MSCs in vivo.
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http://dx.doi.org/10.1016/j.lfs.2016.08.014DOI Listing
October 2016

Hypoxic Preconditioning Improves the Therapeutic Potential of Aging Bone Marrow Mesenchymal Stem Cells in Streptozotocin-Induced Type-1 Diabetic Mice.

Cell Reprogram 2016 10 8;18(5):344-355. Epub 2016 Aug 8.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi , Karachi, Pakistan .

Insulin replacement is the current therapeutic option for type-1 diabetes. However, exogenous insulin cannot precisely represent the normal pattern of insulin secretion. Another therapeutic strategy is transplantation of pancreatic islets, but this is limited by immune rejection, intrinsic complications, and lack of donor availability. Stem cell therapy that results in the regeneration of insulin-producing cells represents an attractive choice. However, with advancing age, stem cells also undergo senescence, which leads to changes in the function of various cellular processes that result in a decrease in the regeneration potential of these aging stem cells. In this study, the effect of young and aging mesenchymal stem cells (MSCs) on the regeneration of pancreatic beta cells in streptozotocin (STZ)-induced type-1 diabetic mice was observed after hypoxic preconditioning. Hypoxia was chemically induced by 2, 4-dinitrophenol (DNP). Plasma insulin and glucose levels were measured at various time intervals, and pancreatic sections were analyzed histochemically. The effect of DNP was also analyzed on apoptosis of MSCs by flow cytometry and on gene expression of certain growth factors by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). We observed that hypoxic preconditioning caused changes in the gene expression levels of growth factors in both young and aging MSCs. Young MSCs showed significant regeneration potential compared with the aging cells in vivo. However, hypoxic preconditioning was able to improve the regeneration potential of aging MSCs. It is concluded from the present study that the regeneration potential of aging MSCs into pancreatic β-cells can be enhanced by hypoxic preconditioning, which causes changes in the gene expression of certain growth factors.
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http://dx.doi.org/10.1089/cell.2016.0002DOI Listing
October 2016

Promoting effect of small molecules in cardiomyogenic and neurogenic differentiation of rat bone marrow-derived mesenchymal stem cells.

Drug Des Devel Ther 2016 24;10:81-91. Epub 2015 Dec 24.

Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

Small molecules, growth factors, and cytokines have been used to induce differentiation of stem cells into different lineages. Similarly, demethylating agents can trigger differentiation in adult stem cells. Here, we investigated the in vitro differentiation of rat bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes by a demethylating agent, zebularine, as well as neuronal-like cells by β-mercaptoethanol in a growth factor or cytokines-free media. Isolated bone marrow-derived MSCs cultured in Dulbecco's Modified Eagle's Medium exhibited a fibroblast-like morphology. These cells expressed positive markers for CD29, CD44, and CD117 and were negative for CD34 and CD45. After treatment with 1 μM zebularine for 24 hours, the MSCs formed myotube-like structures after 10 days in culture. Expression of cardiac-specific genes showed that treated MSCs expressed significantly higher levels of cardiac troponin-T, Nkx2.5, and GATA-4 compared with untreated cells. Immunocytochemical analysis showed that differentiated cells also expressed cardiac proteins, GATA-4, Nkx 2.5, and cardiac troponin-T. For neuronal differentiation, MSCs were treated with 1 and 10 mM β-mercaptoethanol overnight for 3 hours in complete and serum-free Dulbecco's Modified Eagle's Medium, respectively. Following overnight treatment, neuron-like cells with axonal and dendritic-like projections originating from the cell body toward the neighboring cells were observed in the culture. The mRNA expression of neuronal-specific markers, Map2, Nefl, Tau, and Nestin, was significantly higher, indicating that the treated cells differentiated into neuronal-like cells. Immunostaining showed that differentiated cells were positive for the neuronal markers Flk, Nef, Nestin, and β-tubulin.
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http://dx.doi.org/10.2147/DDDT.S89658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699543PMC
October 2016

Transcription profile of genes affected in response to pathological changes in drug-induced rat model of acute kidney injury.

Ren Fail 2015 Aug 26;37(7):1225-31. Epub 2015 Jun 26.

a Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi , Karachi , Pakistan .

Objective: The objective of the present study was to examine the changes in the expression profile of certain genes in rat model of gentamicin-induced acute kidney injury (AKI) and to see whether time period and routes of administration affect their expression levels.

Methods: Rat AKI model was established with gentamicin injection using two different routes of administration and two different time periods. The models were evaluated through histopathological observations. Renal specific genes were selected on the basis of their role during kidney injury. These genes were analyzed through reverse transcriptase (RT) PCR.

Results: Marked disorganization of normal structure of proximal and distal tubules was observed in all the gentamicin-treated groups. Many tubules showed loss of brush border and presence of intratubular protein casts. Changes in gene expression levels were observed for kidney injury molecule (KIM-1), osteopontin, bone morphogenic protein-7 (BMP-7), extracellular signal-regulated kinases (ERK), stem cell factor (SCF) and IL-7 receptor with different levels of significance in the renal injury groups studied depending on the time period and route of administration.

Conclusion: Gene expression seems to be dependent partly on the type of injury, route of administration and time period after induction of injury. An improved mechanistic understanding of gene regulation pathways in AKI may provide basis for potential therapeutic development.
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http://dx.doi.org/10.3109/0886022X.2015.1057801DOI Listing
August 2015

Hypoxic stress and IL-7 gene overexpression enhance the fusion potential of rat bone marrow mesenchymal stem cells with bovine renal epithelial cells.

Mol Cell Biochem 2015 May 10;403(1-2):125-37. Epub 2015 Feb 10.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan.

Transplantation of mesenchymal stem cells (MSCs) has been shown to enhance the improvement in kidney function following injury. However, the poor survival and grafting of the stem cells to the site of injury has restricted their therapeutic efficacy. Accelerated regeneration potential of MSCs has been observed when they were exposed to hypoxic stress or genetic modulation by various cytokines and growth factors. These preconditioning strategies may stimulate endogenous mechanisms resulting in multiple cellular responses. In this study, we used IL-7 gene to transfect MSCs. IL-7 is a hematopoietic growth factor that plays an important role in cell survival, proliferation, and differentiation. MSCs were also subjected to hypoxic stress for 8 and 24 h. These preconditioned MSCs were co-cultured with cisplatin-treated injured Mardin-Darby bovine kidney (MDBK) cells and their fusion potential was analyzed. Flow cytometry of fluorescently labeled preconditioned MSCs and injured MDBK cells revealed evidence of significant (P < 0.001) cell fusion compared to that of the normal MSCs. In addition, we also observed improved migration ability of these preconditioned MSCs in the in vitro wound healing assay, as compared to the normal MSCs. We conclude that hypoxic stress and IL-7 overexpression can enhance the renal regeneration potential of MSCs. This study would help in designing more potent therapeutic strategy in which preconditioned MSCs can be used for renal regeneration.
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http://dx.doi.org/10.1007/s11010-015-2343-0DOI Listing
May 2015

Dinitrophenol modulates gene expression levels of angiogenic, cell survival and cardiomyogenic factors in bone marrow derived mesenchymal stem cells.

Gene 2015 Jan 28;555(2):448-57. Epub 2014 Oct 28.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. Electronic address:

Various preconditioning strategies influence regeneration properties of stem cells. Preconditioned stem cells generally show better cell survival, increased differentiation, enhanced paracrine effects, and improved homing to the injury site by regulating the expression of tissue-protective cytokines and growth factors. In this study, we analyzed gene expression pattern of growth factors through RT-PCR after treatment of mesenchymal stem cells (MSCs) with a metabolic inhibitor, 2,4 dinitrophenol (DNP) and subsequent re-oxygenation for periods of 2, 6, 12 and 24h. These growth factors play important roles in cardiomyogenesis, angiogenesis and cell survival. Mixed pattern of gene expression was observed depending on the period of re-oxygenation. Of the 13 genes analyzed, ankyrin repeat domain 1 (Ankrd1) and GATA6 were downregulated after DNP treatment and subsequent re-oxygenations. Ankrd1 expression was, however, increased after 24h of re-oxygenation. Placental growth factor (Pgf), endoglin (Eng), neuropilin (Nrp1) and jagged 1 (Jag1) were up-regulated after DNP treatment. Gradual increase was observed as re-oxygenation advances and by the end of the re-oxygenation period the expression started to decrease and ultimately regained normal values. Epiregulin (Ereg) was not expressed in normal MSCs but its expression increased gradually from 2 to 24h after re-oxygenation. No change was observed in the expression level of connective tissue growth factor (Ctgf) at any time period after re-oxygenation. Kindlin3, kinase insert domain receptor (Kdr), myogenin (Myog), Tbx20 and endothelial tyrosine kinase (Tek) were not expressed either in normal cells or cells treated with DNP. It can be concluded from the present study that MSCs adjust their gene expression levels under the influence of DNP induced metabolic stress. Their levels of expression vary with varying re-oxygenation periods. Preconditioning of MSCs with DNP can be used for enhancing the potential of these cells for better regeneration.
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http://dx.doi.org/10.1016/j.gene.2014.10.045DOI Listing
January 2015

Conditioned medium enhances the fusion capability of rat bone marrow mesenchymal stem cells and cardiomyocytes.

Mol Biol Rep 2014 May 28;41(5):3099-112. Epub 2014 Jan 28.

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan.

Mesenchymal stem cells (MSCs) show accelerated regeneration potential when these cells experience hypoxic stress. This "preconditioning" has shown promising results with respect to cardio-protection as it stimulates endogenous mechanisms resulting in multiple cellular responses. The current study was carried out to analyze the effect of hypoxia on the expression of certain growth factors in rat MSCs and cardiomyocytes (CMs). Both cell types were cultured and assessed separately for their responsiveness to hypoxia by an optimized dose of 2,4,-dinitrophenol (DNP). These cells were allowed to propagate under normal condition for either 2 or 24 h and then analyzed for the expression of growth factors by RT-PCR. Variable patterns of expression were observed which indicate that their expression depends on the time of re-oxygenation and extent of hypoxia. To see whether the growth factors released during hypoxia affect the fusion of MSCs with CMs, we performed co-culture studies in normal and conditioned medium. The conditioned medium is defined as the medium in which CMs were grown for re-oxygenation till the specified time period of either 2 or 24 h after hypoxia induction. The results showed that the fusion efficiency of cells was increased when the conditioned medium was used as compared to that in the normal medium. This may be due to the presence of certain growth factors released by the cells under hypoxic condition that promote cell survival and enhance their fusion or regenerating ability. This study would serve as another attempt in designing a therapeutic strategy in which conditioned MSCs can be used for ischemic diseases and provide more specific therapy for cardiac regeneration.
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http://dx.doi.org/10.1007/s11033-014-3170-1DOI Listing
May 2014

Sequence analysis and structure prediction of enoyl-CoA hydratase from Avicennia marina: implication of various amino acid residues on substrate-enzyme interactions.

Phytochemistry 2013 Oct 26;94:36-44. Epub 2013 Jun 26.

H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan.

Enoyl-CoA hydratase catalyzes the hydration of 2-trans-enoyl-CoA into 3-hydroxyacyl-CoA. The present study focuses on the correlation between the functional and structural aspects of enoyl-CoA hydratase from Avicennia marina. We have used bioinformatics tools to construct and analyze 3D homology models of A. marina enoyl-CoA hydratase (AMECH) bound to different substrates and inhibitors and studied the residues involved in the ligand-enzyme interaction. Structural information obtained from the models was compared with those of the reported crystal structures. We observed that the overall folds were similar; however, AMECH showed few distinct structural changes which include structural variation in the mobile loop, formation and loss of certain interactions between the active site residues and substrates. Some changes were also observed within specific regions of the enzyme. Glu106 is almost completely conserved in sequences of the isomerases/hydratases including AMECH while Glu86 which is the other catalytic residue in most of the isomerases/hydratases is replaced by Gly and shows no interaction with the substrate. Asp114 is located within 4Å distance of the catalytic water which makes it a probable candidate for the second catalytic residue in AMECH. Another prominent feature of AMECH is the presence of structurally distinct mobile loop having a completely different coordination with the hydrophobic binding pocket of acyl portion of the substrate.
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http://dx.doi.org/10.1016/j.phytochem.2013.05.018DOI Listing
October 2013

CA 15-3 (Mucin-1) and physiological characteristics of breast cancer from Lahore, Pakistan.

Asian Pac J Cancer Prev 2012 ;13(10):5257-61

Department of Biochemistry, Jinnah Medical College, Lahore, Pakistan.

Background: High incidence of breast cancer and its fatal effect has reached an alarming stage across the globe, including the third world countries. Many factors have been reported to be associated with the development of breast cancer but detailed structural and functional information is missing. CA 15-3 is one of the known potential tumor marker of breast cancer; however little is known about structure and functional site of this protein. Present study aims to investigate the functional role of CA 15-3 in breast cancer, especially in development and metastasis.

Material And Methods: Hundred female breast cancer patients confirmed by histopathological reports were included in the study. Their physiological characters were recorded in a performa. Enzyme linked immunosorbent assay (ELISA) technique was used to estimate serum CA 15-3 level. Immunohistochemistry was done for estrogen (ER), progesterone (PR) and Her2/neu receptors expression.

Results: The study revealed the details of physiological characteristics of female breast cancer. Mean age was 37.72 ± 5.99 and 55.05 ± 7.28 years and serum CA 15-3 (MUC1) level was 60.47 ± 8.59 and 63.17 ± 4.58 U/ml in pre and post-menopause respectively, and both groups of women had sedentary life style. Their receptor status especially of progesterone, estrogen and HER-2/neu were positive in 50% of premenopausal women and 65% of postmenopausal women.

Conclusion: There are multiple physiological factors promoting breast cancer. High serum CA 15-3 level and hormonal imbalance of ER, PR and Her2/neu appears to be the main cause of breast cancer. It may be possible that the functional sites of these proteins may be altered which may increase the chances of metastasis in breast cancer.
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http://dx.doi.org/10.7314/apjcp.2012.13.10.5257DOI Listing
June 2013

DNA methylation inhibitors, 5-azacytidine and zebularine potentiate the transdifferentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes.

Cardiovasc Ther 2013 Aug;31(4):201-9

Dr. Panjwani Center for Molecular Medicine and Drug Research (PCMD), International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi, Pakistan.

Background: Mesenchymal stem cells (MSCs) have immense self-renewal capability. They can be differentiated into many cell types and therefore hold great potential in the field of regenerative medicine. MSCs can be converted into beating cardiomyocytes by treating them with DNA-demethylating agents. Some of these compounds are nucleoside analogs that are widely used for studying the role of DNA methylation in biological processes as well as for the clinical treatment of leukemia and other carcinomas.

Aims: To achieve a better therapeutic option for cardiovascular regeneration, this study was carried out using MSCs treated with two synthetic compounds, zebularine and 5-azacytidine. It can be expected that treated MSCs prior to transplantation may increase the likelihood of successful regeneration of damaged myocardium.

Methods: The optimized concentrations of these compounds were added separately into the culture medium and the treated cells were analyzed for the expression of cardiac-specific genes by RT-PCR and cardiac-specific proteins by immunocytochemistry and flow cytometry. Treated MSCs were cocultured with cardiomyocytes to see the fusion capability of these cells.

Results: mRNA and protein expressions of GATA4, Nkx2.5, and cardiac troponin T were observed in the treated MSCs. Coculture studies of MSCs and cardiomyocytes have shown improved fusion with zebularine-treated MSCs as compared to untreated and 5-azacytidine-treated MSCs.

Conclusion: The study is expected to put forth another valuable aspect of certain compounds, that is, induction of transdifferentiation of MSCs into cardiomyocytes. This would serve as a tool for modified cellular therapy and may increase the probability of better myocardial regeneration.
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http://dx.doi.org/10.1111/j.1755-5922.2012.00320.xDOI Listing
August 2013

Predicting the functionally distinct residues in the heme, cation, and substrate-binding sites of peroxidase from stress-tolerant mangrove specie, Avicennia marina.

Cell Stress Chaperones 2011 Nov 10;16(6):585-605. Epub 2011 Jun 10.

H.E.J. Research Institute of Chemistry, University of Karachi, Pakistan.

Recent work was conducted to predict the structure of functionally distinct regions of Avicennia marina peroxidase (AP) by using the structural coordinates of barley grains peroxidase as the template. This enzyme is utilized by all living organisms in many biosynthetic or degradable processes and in defense against oxidative stress. The homology model showed some distinct structural changes in the heme, calcium, and substrate-binding regions. Val53 was found to be an important coordinating residue between distal calcium ion and the distal heme site while Ser176 is coordinated to the proximal histidine through Ala174 and Leu172. Different ionic and hydrogen-bonded interactions were also observed in AP. Analyses of various substrate-enzyme interactions revealed that the substrate-binding pocket is provided by the residues, His41, Phe70, Gly71, Asp138, His139, and Lys176; the later three residues are not conserved in the peroxidase family. We have also performed structural comparison of the A. marina peroxidase with that of two class III salt-sensitive species, peanut and soybean. Four loop regions were found to have largest structural deviation. The overall protein sequence was also analyzed for the presence of probable post-translational modification sites and the functional significance of these sites were outlined.
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http://dx.doi.org/10.1007/s12192-011-0269-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220393PMC
November 2011

Granzyme M: characterization with sites of post-translational modification and specific sites of interaction with substrates and inhibitors.

Mol Biol Rep 2011 Jun 28;38(5):2953-60. Epub 2010 Jan 28.

Department of Biochemistry, Fatima Jinnah Medical College, Lahore, Pakistan.

Granzymes kill cells in a variety of ways. They induce mitochondrial dysfunction through caspase dependent and caspase-independent pathways and destroy DNA and the integrity of the nucleus. For gaining a better understanding of the molecular function of granzyme M and its NK cell specificity, structural characterization of this enzyme by molecular modeling as well as its detailed comparison with other granzymes is presented in this study. The study includes mode of action of granzyme M using cationic binding sites, substrate specificity, post-translational structural modification and its functional relationship and interaction of the enzyme with inhibitor in an attempt to explore how the activity of human granzyme M is controlled under physiological conditions. It is concluded from the present study that the post-translational modification, including Oglycosylation of serine, phosphorylation of serine and threonine and myristoylation of glycine, play an important role in the interaction of enzyme with the cell surface membrane and regulate protein trafficking and stability. Phosphorylated serine and threonine also plays a role in tumor elimination, viral clearance and tissue repair. In Gzm M there are cationic sites, cs1 and cs2 that may participate in binding of Gzm M to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm. Gzm M shows apoptotic activity both by caspase dependent and independent pathways. Modeling of inhibitors bound to the granzyme active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft.
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http://dx.doi.org/10.1007/s11033-010-9959-7DOI Listing
June 2011

Expression and activity of paraoxonase 1 in human cataractous lens tissue.

Free Radic Biol Med 2009 Apr 23;46(8):1089-95. Epub 2009 Jan 23.

Department of Biochemistry, University of Karachi, Karachi, Pakistan.

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that is believed to be involved in the protection against oxidative stress. There is evidence that paraoxonase activity is reduced in patients with diabetes and cataract. In the current study, we analyzed mRNA expression of PON1 as well as other members of the paraoxonase family, PON2 and PON3, in human cataractous lens samples. Our results indicate that only PON1 is expressed at the gene and protein levels in human lens tissues. We quantified MDA levels and measured PON1 (paraoxonase/arylesterase) enzymatic activities in subjects suffering from cataract due to aging and diabetes. Decreased PON1 activity was more pronounced in diabetic patients (p< 0.001) compared to senile subjects, which may be due to glycation and increased oxidative insult. To examine the structural alterations that occur in response to glycation, we constructed a three-dimensional model of PON1 and its glycated variant. Glycation at Lys70 and Lys75 is predicted to cause hindrance in binding of substrate to the active site of the enzyme.
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http://dx.doi.org/10.1016/j.freeradbiomed.2009.01.012DOI Listing
April 2009

Advanced glycation end-products in senile diabetic and non-diabetic patients with cardiovascular complications.

Age (Dordr) 2008 Dec 3;30(4):303-9. Epub 2008 Sep 3.

Department of Biochemistry, Ziauddin University, 4/B Shahra-e- Ghalib, Clifton, Karachi, 75600, Pakistan.

Advanced glycation end products (AGEs) have been reported to contribute to aging and cardiovascular complications. In the present study, the immunoreactivity of AGEs in human serum samples of healthy older subjects (n = 31), senile diabetic patients without cardiovascular complications (n = 33), senile diabetic patients with cardiovascular complications (n = 32), senile non-diabetic patients with cardiovascular complications (n = 30) ,and healthy young subjects (n = 31) were investigated. The patients were selected on clinical grounds from the National Institute of Cardiovascular Disease, Karachi and the Jinnah Postgraduate Medical Centre, Karachi, Pakistan. Fasting blood glucose, HbA(1C) and serum fructosamine levels were significantly (P < 0.001) increased in senile diabetic patients with and without cardiovascular complications as compared to non-diabetic senile patients with cardiovascular complications and healthy older subjects. Additionally, serum AGEs were found to be significantly (P < 0.001) increased in senile diabetic patients with cardiovascular complications and senile non-diabetic patients with cardiovascular complications, followed by diabetic patients without cardiovascular complications as compared to healthy older subjects and young control subjects. However, no significant difference was found in the senile diabetic patients without cardiovascular complications and senile non-diabetic patients with cardiovascular complications. In contrast to all four senile groups, serum AGEs were significantly (P < 0.001) lower in young control subjects. The AGEs distribution in the senile groups corroborates the hypothesis that the advanced glycation process might play a role in the development of cardiovascular complications, which are more severe in diabetic patients compared with non-diabetic patients with cardiovascular complications.
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http://dx.doi.org/10.1007/s11357-008-9072-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2585643PMC
December 2008

Could oxidative stress associate with age products in cataractogenesis?

Curr Eye Res 2008 Aug;33(8):669-75

Department of Biochemistry, Ziauddin University, Shahrah-e-Ghalib, Clifton, Karachi, Pakistan.

Background: Oxidative stress has been reported to contribute to aging and cataract formation in the lens. The aim was to determine the association of oxidative stress with advanced glycation end products (AGEs) in elderly diabetic and non-diabetic patients with cataract.

Methods: In the present study, malondialdehyde, vitamin E, serum AGEs, and glycemic control were investigated. The study included 156 subjects. Out of them, 30 were normal elderly subjects, 31 were elderly diabetic patients without cataract, 33 were elderly diabetic patients with cataract, 32 were elderly non-diabetic with cataract, and 30 were normal young subjects. The patients were selected on clinical grounds from Eye Ward, Jinnah Postgraduate Medical Centre, Karachi, Pakistan.

Results: Positive significant correlation was observed between s-AGEs and malondialdehyde in elderly diabetic and non-diabetic patients with cataract. Negative significant correlation was observed between s-AGEs and vitamin E in elderly diabetic and non-diabetic patients with cataract. However, the malondialdehyde and serum AGEs were found to be significantly increased (p < 0.001) in elderly diabetic and non-diabetic patients with and without cataract compared with elderly control subjects. In contrast to all four senile groups, the serum AGEs was significantly lower (p < 0.001) in young control subjects. Serum vitamin E was found to be significantly decreased (p < 0.001) in elderly diabetic patients with and without cataract compared with elderly control subjects. Fasting blood glucose, HbA(1C) and serum fructosamine levels were significantly increased (p < 0.001) in elderly diabetic patients with and without cataract compared with non-diabetic elderly patients with cataract and elderly control subjects.

Conclusions: This study revealed that increased AGEs were associated with oxidative stress in the elderly groups. AGE, as a result of oxidative stress, might have a role in cataract formation, which, in diabetic patients, occurs vigorously as compared with non-diabetic cataract patients.
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http://dx.doi.org/10.1080/02713680802250939DOI Listing
August 2008

Advanced glycation end products in senile diabetic and nondiabetic patients with cataract.

J Diabetes Complications 2009 Sep-Oct;23(5):343-8. Epub 2008 May 27.

Department of Biochemistry, Ziauddin University, Shahrah-e-Ghalib, Clifton, Karachi-75600, Pakistan.

Background: Advanced glycation end products (AGE) have been reported to contribute to aging and cataract formation in the lens. In the present study, AGE immunoreactivity in human serum samples of normal senile subjects (n=31), senile diabetic patients without cataract (n=33), senile diabetic patients with cataract (n=30), senile nondiabetic with cataract (n=30), and normal young subjects (n=31) was investigated.

Methods: A noncompetitive ELISA with polyclonal anti-AGE antibody was performed. The patients were selected on clinical grounds from Eye Ward, Jinnah Postgraduate Medical Centre, Karachi, Pakistan.

Results: Fasting blood glucose, glycosylated hemoglobin, and serum fructosamine were estimated. Fasting blood glucose, HbA(1C), and serum fructosamine levels were significantly (P<.001) increased in senile diabetic patients with and without cataract as compared to nondiabetic senile patients with cataract and senile control subjects. However, the serum AGEs were found to be significantly (P<.001) increased in senile diabetic patients with cataract and senile nondiabetic patients with cataract followed by the diabetic patients without cataract as compared to senile control and young control subjects. In contrast to all four senile groups, the serum AGEs were significantly (P<.001) lower in young control subjects.

Conclusions: The AGE distribution in the senile groups corroborates the hypothesis that the advanced glycation process might have a role in cataract formation, which in diabetic patients occurs vigorously as compared with nondiabetic cataract patients.
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http://dx.doi.org/10.1016/j.jdiacomp.2008.04.001DOI Listing
January 2010

Transcriptional profiling of young and old mesenchymal stem cells in response to oxygen deprivation and reparability of the infarcted myocardium.

J Mol Cell Cardiol 2008 Mar 7;44(3):582-96. Epub 2007 Dec 7.

Department of Pathology and Laboratory Medicine, 231-Albert Sabin Way, University of Cincinnati, OH 45267-0529, USA.

Most clinical studies have used autologous bone marrow (BM) stem cells for myocardial regeneration in elderly patients. We hypothesize that aging impairs the survival and differentiation potential of BM stem cells thus limiting their therapeutic efficacy. BM-derived MSCs from young ((Yng)MSCs; 8-12 weeks) and old ((Old)MSCs; 24-26 months) rats were purified and assessed for their responsiveness to anoxia and reparability of infarcted heart. Higher expression of angiogenic growth factors was observed by (Yng)MSCs under anoxia as compared to (Old)MSCs, cultured either alone or in co-culture ((Co-old)MSCs) with (Yng)MSCs. Likewise, (Yng)MSCs were more tolerant to apoptotic stimuli and showed higher ability to form tubular structures during in vitro Matrigel assay as compared to (Old)MSCs and (Co-old)MSCs with a possible role of p21 and p27 as contributory survival factors. For in vivo studies, acute myocardial infarction model was developed in Fischer-344 rats (n=38). The animals were grouped to receive 70 microl basal DMEM without cells (group 1) or containing 2 x 10(6)(Yng)MSCs (PKH67 labeled; group 2) or (Old)MSCs (PKH26 labeled; group 3) and mixture of (Yng)MSCs + (Old)MSCs (1 x 10(6) cells each; group 4). Histological studies revealed that by day 7, (Yng)MSCs showed elongated morphology with orientation similar to the host muscle architecture. Electron microscopy and confocal imaging after fluorescent immunostaining showed superior angiomyogenic potential of (Yng)MSCs. Echocardiography showed significantly preserved heart function indices in the animals transplanted with (Yng)MSCs. Aging impairs the responsiveness of (Old)MSCs to anoxia and their differentiation potential. (Yng)MSCs fail to alter the survival of (Old)MSCs under in vitro as well as in vivo conditions. It is therefore concluded that transplantation of stem cells from young donors would be a better option for heart cell therapy in future clinical studies.
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http://dx.doi.org/10.1016/j.yjmcc.2007.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753254PMC
March 2008

Supportive interaction between cell survival signaling and angiocompetent factors enhances donor cell survival and promotes angiomyogenesis for cardiac repair.

Circ Res 2006 Sep 7;99(7):776-84. Epub 2006 Sep 7.

Department of Pathology and Laboratory Medicine, 231 Albert Sabin Way, University of Cincinnati, OH 45267-0529, USA.

Akt is a major cell survival and angiogenic mediator downstream of angiopoietin-1 (Ang-1)/Tie-2 signaling pathway. We hypothesize that transplantation of mesenchymal stem cells (MSCs) co-overexpressing Ang-1 and Akt lead to better prognosis. Ang-1 and Akt genes were adenovirally transduced into MSCs from male Fischer rats. Cytoprotective effects of transgene overexpression in vitro were assessed by exposure of cells to 8 hours of anoxia. TUNEL and measurement of lactate dehydrogenase showed that MSCs co-overexpressing Ang-1 and Akt (MAAs) were more resistant to anoxia as compared with the nontransduced MSCs or those transduced with Ang-1 or Akt alone. For in vivo studies, after permanent coronary artery occlusion, animals were grouped (n=20/group) to receive intramyocardial injections of 70 microL of basal medium without cells (group 1) or containing 3x10(6) nontransduced MSCs (group 2) or MAAs (group 3). Four animals per group were euthanized on 4, 7, and 14 days after cell transplantation for molecular studies. Extensive survival of MAAs was observed in group 3, which continued to co-overexpress transgenes in rat heart at 2 weeks after cell transplantation. Immunohistology at 4 weeks revealed myogenic differentiation of donor cells at the site of cell graft. Blood vessel density was highest in the infarct and periinfarct regions in group 3 (P<0.05). Echocardiography at 4 weeks showed that heart function indices were significantly improved in group 3 (P<0.05), including ejection fraction and fractional shortening as compared with groups 1 and 2. We conclude that supportive interaction between Ang-1 and Akt during MSC transplantation gave better prognosis via enhanced cell survival, improved angiomyogenesis, and restored global cardiac function.
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http://dx.doi.org/10.1161/01.RES.0000244687.97719.4fDOI Listing
September 2006

Injected tryptophan increases brain but not plasma tryptophan levels more in restrained rats.

Pak J Pharm Sci 2003 Jan;16(1):51-7

Department of Biochemistry, University of Karachi, Karachi-75270, Pakistan.

Immobilization stress given for one hour decreased cumulative food intake and growth rate in rats. Activity in an open field scored next day was also smaller in the restrained animals. Stress is known to enhance brain serotonin (5-hydroxytryptamine; 5-HT) metabolism. It is however not clear, whether this enhancement is caused by an increase in the activity of tryptophan hydroxylase, the rate limiting enzyme of 5-HT biosynthesis or availability of the precursor tryptophan to the brain. In order to determine the utilization of tryptophan via serotonin path way in brain during stress; rats were injected with saline or tryptophan (50 mg/kg, i.p.). A group of both saline and tryptophan injected animals was restrained for one hour, while another group left unrestrained. The animals were killed immediately after the termination of stress period to collect plasma and brain samples. Injected tryptophan resulted in 3-4 fold rise of plasma tryptophan in both unstressed and stressed animals in one hour. Stress alone however had no effect on plasma total tryptophan concentration. Free tryptophan in plasma, brain tryptophan, 5-HT and 5-hydroxyindole acetic acid (5-HIAA) all increased by both stress and tryptophan injection. The increases of brain tryptophan, 5-HT and 5-HIAA but not of free tryptophan in plasma were considerably greater in restrained animals given tryptophan load. The results suggest that enhancement of brain serotonin metabolism during stress is caused by an increase in the availability of tryptophan to the brain as well as an increase in the activity of the enzyme tryptophan hydroxylase. Factors affecting the availability of tryptophan to the brain are discussed.
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January 2003

Three-dimensional structure prediction of bovine AP lyase, BAP1: prediction of interaction with DNA and alterations as a result of Arg176-->Ala, Asp282-->Ala, and His308-->Asn mutations.

Biochem Biophys Res Commun 2005 Jan;326(4):711-7

H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan.

BAP1 is an apurinic/apyrimidinic lyase (AP lyase) that plays an important role in the repair of DNA damage. The present study deals with the prediction of the 3D structure of bovine AP lyase based on its sequence homology with human AP lyase. The predicted 3D model of bovine AP1 shows remarkable similarity with human endonuclease in the overall 3D fold. However, significant differences in the model and the X-ray structure were located at some of the important sites. We have analyzed the active center of the enzyme and other sites that are involved in DNA repair. A number of amino acids bind the bases located in the major/minor grooves of DNA. An insertion of Arg176 in the major groove and Met270 in the minor groove caps the DNA bound enzyme's active site, stabilizing the extra helical AP site conformation and effectively locking the protein onto the AP-DNA. Three BAP1 mutants were also modeled and analyzed as regards the changes in the structure. Substitution of Arg176-->Ala leads to the loss of DNA binding whereas mutation of Asp282-->Ala and His308-->Asn leads to a decrease in the enzymatic activity.
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http://dx.doi.org/10.1016/j.bbrc.2004.11.103DOI Listing
January 2005

Modeling the mutational effects on calmodulin structure: prediction of alteration in the amino acid interactions.

Biochem Biophys Res Commun 2004 Apr;317(2):363-9

Department of Pathology, Fatima Jinnah Medical College, Lahore, Pakistan.

Calmodulin (CaM) is a highly conserved 17kDa eukaryotic protein that can bind specifically to over 100 protein targets in response to a Ca2+ signal. Present study was planned to mutate the crucial residues of N-terminal lobe, central helix, and C-terminal lobe that play important roles in activating and binding of enzymes. In all, 10 mutations were carried out in the predicted 3D structure of calmodulin using the computer program MODELLER 6v2. Mutations at specific residues in both the N-terminal and C-terminal regions resulted in the change in the interaction pattern of these amino acids. No significant change was however predicted by mutating amino acid residues in the central helix. The predicted alteration in the interaction of specific amino acids may either alter the binding affinity with calcium ions or decrease the ability of calmodulin to activate the specific enzymes.
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http://dx.doi.org/10.1016/j.bbrc.2004.03.051DOI Listing
April 2004