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

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.

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

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.

N-(2-hydroxy phenyl) acetamide: a novel suppressor of Toll-like receptors (TLR-2 and TLR-4) in adjuvant-induced arthritic rats.

Toll-like receptors (TLRs) are key recognition structures of immune system and recently emerged as potential contributors to the inflammation observed in human and rodent models of arthritis. Present study aims to investigate the effect of N-(2-hydroxy phenyl)-acetamide (NA-2) on modulation of TLRs in the development of adjuvant-induced arthritis. Arthritis was induced by intradermal administration of heat-killed Mycobacterium tuberculosis H37Ra. The treatment of NA-2 (5 mg/kg) and indomethacin (5 mg/kg) was started in their respective group on the day of arthritis induction. Body weights, paw volume measurements, and nociception sensation (Plantar test) were done on alternate days to monitor the progression of the disease until arthritis score of four was observed in arthritic control group. Along with the clinical signs, histopathology of knee joints was also performed. The splenocytes cultures were prepared from each group; TLR-2 and TLR-4 mRNAs were analyzed in 48-h cultured splenocytes using RT-PCR; and the supernatants were used to determine IL-1ß and TNF-α by ELISA. A significant reversal of deficit seen in body weights of the arthritic control group was observed in NA-2-treated animals with a parallel decrease in paw edema and transmission of nociception. Remission of the clinical signs and nociception was associated with improved histology. Compared with arthritic control, NA-2 treatment significantly decreased the level of IL-1ß (p < 0.003) and TNF-α (p < 0.001) in the supernatants of cultured splenocytes. Likewise, NA-2 also reduced the expression of TLRs mRNA. Our findings suggest that NA-2 affects AIA in a pleiotropic manner, suppressing TLRs-mediated joint inflammation and related symptoms.

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

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.

N-(2-hydroxy phenyl) acetamide produces profound inhibition of c-Fos protein and mRNA expression in the brain of adjuvant-induced arthritic rats.

Chronic pain and cognitive decline are characteristic symptoms of rheumatoid arthritis. One of the immediate early gene c-fos is overexpressed during peripheral and central noxious conditions and can be used as a marker for neuronal activity/excitability. In the adjuvant-induced arthritis Sprague-Dawley rat model, we examined the dynamics of c-Fos protein and mRNA expression in the amygdala, cortex, hippocampus, and thalamus and evaluated the effects of N-(2-hydroxy phenyl) acetamide (NA-2), a derivative of salicylic acid. The paw volume was assessed as an indicator of peripheral edema and the hyperalgesia associated with arthritis was monitored by gait analysis. The region of interests of the brain from arthritic and non-arthritic animals were used to isolate the RNA and were then reverse transcribed into cDNA. The PCR products were electrophoresed on 1% agarose gel and the gels were visualized in gel-doc system. The frozen brain sections were stained for c-Fos using immunohistochemistry. Negative control experiments were performed without the primary and secondary antibodies to rule out the nonspecific tissue binding of antibodies. We report a significant increase in the c-Fos expression in the arthritic control animals. In comparison to the control group, the treatment of NA-2 treatment was found to block the development of the arthritis-induced c-Fos protein and mRNA expression and peripheral edema. It also significantly reduces the gait deficits which were otherwise observed in the arthritic control group. Both the immunohistochemistry and PCR analysis revealed NA-2 to be more potent in comparison to member of non-steroidal anti-inflammatory drug.

N-(2-hydroxyphenyl)acetamide (NA-2) and Temozolomide synergistically induce apoptosis in human glioblastoma cell line U87.

BACKGROUND: Despite the modern therapies available for treating glioblastoma multiforme (GBM), it is still a deadly disease. The development of new therapeutic strategies for the management of gliomas is therefore crucial. The present study is designed to analyze the therapeutic potentials of synthetic compound N-(2-hydroxyphenyl)acetamide (NA-2) in the treatment of GBM as a single agent or in combination with Temozolomide (TMZ) on glioblastoma cells. METHODS: MTT and TUNEL assays were used to detect the growth inhibitory effect and apoptotic activity of NA-2 alone and in combination with TMZ. Synergy was assessed using combination Index method. The expression of apoptosis related markers Bax, Bcl-2 and caspase-3 were assessed by RT-PCR, whereas, the active caspase-3 protein expression was determined using imunocytochemistry. RESULTS: Both NA-2 and TMZ inhibited the growth of U87 in a dose dependent manner. The combine administration of NA-2 (0.33 mM) and temozolomide (0.1 mM) significantly enhanced the cell growth inhibition and apoptosis. Furthermore RT-PCR and imunocytochemistry data revealed that cooperative apoptosis induction was associated with increased ratio of Bax to Bcl-2 and active Caspase-3 expression. CONCLUSION: Our findings support that NA-2 possesses strong apoptotic activity and the combined administration of NA-2 and TMZ may be therapeutically exploited for the management of GBM.

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

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.

Suppression of c-Fos protein and mRNA expression in pentylenetetrazole-induced kindled mouse brain by isoxylitones.

An early immediate gene c-fos has been proposed as the gene responsible for turning on molecular events that might underlie the long-term neural changes occurring during kindling. We have evaluated the effects of novel anticonvulsant isomeric compounds isoxylitones [(E/Z)-2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine] on the c-Fos protein and mRNA expression in the brain samples of kindled mice and compared it with the normal and untreated kindled groups. Kindling was induced in male NMRI mice by repeated administration of sub-convulsive dose (50 mg/kg) of pentylenetetrazole (PTZ) until a seizure score of 4-5 was achieved. The c-Fos expression was quantified by combination of immunohistochemistry and RT-PCR protocols. Both the immunohistochemical and RT-PCR analysis revealed a marked increase in the expression of c-fos mRNA and protein in the brain regions tested in case of PTZ-kindled control group compared to normal control. In contrast, the isoxylitone (30 mg/kg)-treated group demonstrated significant reduction of c-Fos expression compared to PTZ-kindled control animals. However, low expression of c-fos mRNA was only detected in the thalamus of the isoxylitone-treated brain samples. Based on these observations, we suggest that isoxylitones may have the capacity to control the seizure pattern by mechanism such as the suppression of c-Fos protein and mRNA levels in different regions of the brain. Further investigations to explore the mechanism of action of these compounds are under process.

N-(2-hydroxy phenyl) acetamide inhibits inflammation-related cytokines and ROS in adjuvant-induced arthritic (AIA) rats.

The present study was carried out to study the anti-arthritic and anti-inflammatory activity of N-(2-hydroxy phenyl) acetamide in adjuvant-induced arthritis in adult female Sprague Dawley rats. During experimental period, body weight and paw oedema volume were observed. At the end of each experiment, plasma and serum samples were collected and used for estimation of pro-inflammatory cytokines IL-1 beta and TNF-alpha and oxidative stress markers i.e., nitric oxide, peroxide and GSH. Our results suggested that, the reduction in body weight and increase in paw oedema volume were significantly retarded in the AIA rats receiving 5mg/kg and 10mg/kg doses of N-(2-hydroxy phenyl) acetamide as compared to diseased control animals. The serum levels of IL-1 beta and TNF-alpha were reduced as compared to those in the diseased control group. Treatment with N-(2-hydroxy phenyl) acetamide also altered oxidative stress markers in relation to its anti-inflammatory activity. Based on our results, it can be concluded that N-(2-hydroxy phenyl) acetamide possesses promising anti-arthritic property.