Bharath Chelluboina

Glioma stem cell invasion through regulation of the interconnected ERK, integrin α6 and N-cadherin signaling pathway

The recent characterization of glioma stem cells (GSCs) prompts a necessary examination of the signaling pathways that facilitate invasiveness. Molecular crosstalk between expression mechanisms has been identified in a range of cancers, including glioblastoma multiforme. However, hardly any literature exists that addresses whether cancer stem cells utilize these same interconnected pathways. Protein factors commonly implicated in malignant tumors include extracellular signal-regulated kinase (ERK), N-cadherin, and integrin α6. Although studies have reported the molecular crosstalk involved among these proteins, the present study illustrates the importance of the ERK transduction pathway in N-cadherin and integrin α6 regulated invasion in GSCs. Conversely, the data also suggests that GSCs rely on N-cadherin and integrin α6 interaction to regulate ERK signaling. Moreover, confocal visualization revealed the co-localization of N-cadherin and integrin α6 in GSCs and clinical surgical biopsies extracted from glioma patients. Interestingly, ERK knockdown reduced this co-localization. Upon co-culturing GSCs with human umbilical cord blood stem cells (hUCBSCs), we observed a subsequent decrease in pERK, N-cadherin and integrin α6 expression. In addition, co-culturing hUCBSCs with GSCs decreased co-localization of N-cadherin and integrin α6 in GSCs. Our results demonstrate the dynamic interplay among ERK, N-cadherin and integrin α6 in GSC invasion and also reveal the therapeutic potential of hUCBSCs in treating the molecular crosstalk observed in GSC-regulated invasion.

Temporal Regulation of Apoptotic and Anti-apoptotic Molecules After Middle Cerebral Artery Occlusion Followed by Reperfusion

A tremendous effort has been expended to elucidate the role of apoptotic molecules in ischemia. However, many agents that target apoptosis, despite their proven efficacy in animal models, have failed to translate that efficacy and specificity in clinical settings. Therefore, comprehensive knowledge of apoptotic mechanisms involving key apoptotic regulatory molecules and the temporal expression profiles of various apoptotic molecules after cerebral ischemia may provide insight for the development of better therapeutic strategies aimed at cerebral ischemia. The present study investigates the extent of apoptosis and the regulation of apoptotic molecules both at mRNA and protein levels at various time points after focal cerebral ischemia in a rat model of middle cerebral artery occlusion. In this study, we performed various techniques, such as TTC (2,3,5-triphenyltetrazolium chloride), H&E (hematoxylin and eosin), and TUNEL (terminal deoxy nucleotidyl transferase-mediated nick-end labeling) staining, along with polymerase chain reaction (PCR) microarray, antibody microarray, reverse transcription (RT)-PCR, immunofluorescence, and immunoblot analyses. Our research provided a large list of pro-apoptotic and anti-apoptotic molecules and their temporal expression profiles both at the mRNA and protein levels. This information could be very useful for designing future stroke therapies and aid in targeting the right molecules at critical time to obtain maximum therapeutic benefit.

Post-transcriptional inactivation of matrix metalloproteinase-12 after focal cerebral ischemia attenuates brain damage

This study highlights the possible pathological role of MMP-12 in the context of ischemic stroke. Male rats were subjected to a two-hour middle cerebral artery occlusion (MCAO) procedure. MMP-12 shRNA expressing plasmid formulation was administered to these rats twenty-four hours after reperfusion. The results showed a predominant upregulation of MMP-12 (approximately 47, 58, 143, and 265 folds on days 1, 3, 5, 7 post-ischemia, respectively) in MCAO subjected rats. MMP-12 expression was localized to neurons, oligodendrocytes and microglia, but not astrocytes. Transcriptional inactivation of MMP-12 significantly reduced the infarct size. The percent infarct size was reduced from 62.87 ± 4.13 to 34.67 ± 5.39 after MMP-12 knockdown compared to untreated MCAO subjected rats. Expression of myelin basic protein was increased, and activity of MMP-9 was reduced in ischemic rat brains after MMP-12 knockdown. Furthermore, a significant reduction in the extent of apoptosis was noticed after MMP-12 knockdown. TNFα expression in the ipsilateral regions of MCAO-subjected rats was reduced after MMP-12 knockdown in addition to the reduced protein expression of apoptotic molecules that are downstream to TNFα signaling. Specific knockdown of MMP-12 after focal cerebral ischemia offers neuroprotection that could be mediated via reduced MMP-9 activation and myelin degradation as well as inhibition of apoptosis.

Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration

BackgroundSrc tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration.MethodsMMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis.ResultsImmunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR.ConclusionsTaken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin and iNOS levels.

MMP-12, a Promising Therapeutic Target for Neurological Diseases

The role of matrix metalloproteinase-12 (MMP-12) in the pathogenesis of several inflammatory diseases such as chronic obstructive pulmonary disease, emphysema, and asthma is well established. Several new studies and recent reports from our laboratory and others highlighted the detrimental role of MMP-12 in the pathogenesis of several neurological diseases. In this review, we discuss in detail the pathological role of MMP-12 and the possible underlying molecular mechanisms that contribute to disease pathogenesis in the context of central nervous system diseases such as stroke, spinal cord injury, and multiple sclerosis. The available information on the specific MMP-12 inhibitors used in several preclinical and clinical studies is also reviewed. Based on the reported studies to date, MMP-12 suppression could emerge as a promising therapeutic target for several CNS diseases that were discussed in this review.

Mesenchymal Stem Cell Treatment Prevents Post-Stroke Dysregulation of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases

Background/Aims: Stem cell treatment is one of the potential treatment options for ischemic stroke. We recently demonstrated a protective effect of human umbilical cord blood-derived mesenchymal stem cells (HUCB-MSCs) in a rat model of ischemic stroke. The treatment attenuated apoptosis and prevented DNA damage. A collection of published studies, including several from our laboratory, indicated the induction and detrimental role for several matrix metalloproteinases (MMPs) in post-stroke brain injury. We hypothesized that the HUCB-MSCs treatment after focal cerebral ischemia prevents the dysregulation of MMPs and induces the expression of endogenous tissue inhibitors of metalloproteinases (TIMPs) to neutralize the elevated activity of MMPs. Methods: To test our hypothesis, we administered HUCB-MSCs (0.25 million cells/animal and 1 million cells/animal) intravenously via tail vein to male Sprague-Dawley rats that were subjected to a transient (two-hour) right middle cerebral artery occlusion (MCAO) and one-day reperfusion. Ischemic brain tissues obtained from various groups of rats seven days after reperfusion were subjected to real-time PCR, immunoblot, and immunofluorescence analysis. Results: HUCB-MSCs treatment prevented the induction of MMPs, which were upregulated in ischemia-induced rats that received no treatment. HUCB-MSCs treatment also prevented the induction of TIMPs expression. The extent of prevention of MMPs and TIMPs induction by HUCB-MSCs treatment is similar at both the doses tested. Conclusion: Prevention of stroke-induced MMPs upregulation after HUCB-MSCs treatment is not mediated through TIMPs upregulation.

Transdifferentiation of differentiated stem cells contributes to remyelination.

Evidence suggests that transdifferentiation of mesenchymal stem cells (MSCs) into various neuronal cells contributes to functional recovery after experimental spinal cord injury. Qiu et al. have recently published an exciting article in Stem Cell Research & Therapy demonstrating the transdifferentiation of already differentiated MSCs that contributes to remyelination of injured/regenerating axons, and thereby to functional recovery of spinal cord injured animals. The authors highlight the importance of interaction between neurotrophin-3 and tropomyosin receptor kinase C for the observed effects. This study provided important evidence that manipulation of rat bone marrow-derived MSCs before transplantation could enhance the therapeutic benefit of cell-based treatment.

Post-stroke mRNA expression profile of MMPs: effect of genetic deletion of MMP-12

Background and purpose Recent reports from our laboratory demonstrated the post-ischaemic expression profile of various matrix metalloproteinases (MMPs) in rats and the detrimental role of MMP-12 in post-stroke brain damage. We hypothesise that the post-stroke dysregulation of MMPs is similar across species and that genetic deletion of MMP-12 would not affect the post-stroke expression of other MMPs. We tested our hypothesis by determining the pre-ischaemic and post-ischaemic expression profile of MMPs in wild-type and MMP-12 knockout mice. Methods Focal cerebral ischaemia was induced in wild-type and MMP-12 knockout mice by middle cerebral artery occlusion procedure by insertion of a monofilament suture. One hour after ischaemia, reperfusion was initiated by removing the monofilament. One day after reperfusion, ischaemic brain tissues from various groups of mice were collected, and total RNA was isolated and subjected to cDNA synthesis followed by PCR analysis. Results Although the post-stroke expression profile of MMPs in the ischaemic brain of mice is different from rats, there is a clear species similarity in the expression of MMP-12, which was found to be predominantly upregulated in both species. Further, the post-stroke induction or inhibition of various MMPs in MMP-12 knockout mice is different from their respective expression profile in wild-type mice. Moreover, the brain mRNA expression profile of various MMPs in MMP-12 knockout mice under normal conditions is also different to their expression in wild-type mice. Conclusions In the ischaemic brain, MMP-12 upregulates several fold higher than any other MMP. Mice derived with the genetic deletion of MMP-12 are constitutive and have altered MMP expression profile both under normal and ischaemic conditions.

Prevention of the Severity of Post-ischemic Inflammation and Brain Damage by Simultaneous Knockdown of Toll-like Receptors 2 and 4

Toll-like receptor 2 (TLR2) and TLR4 belong to a family of highly conserved pattern recognition receptors and are well-known upstream sensors of signaling pathways of innate immunity. TLR2 and TLR4 upregulation is thought to be associated with poor outcome in stroke patients. We currently show that transient focal ischemia in adult rats induces TLR2 and TLR4 expression within hours and shRNA-mediated knockdown of TLR2 and TLR4 alone and in combination decreases the infarct size and swelling. We further show that TLR2 and TLR4 knockdown also prevented the induction of their downstream signaling molecules MyD88, IRAK1, and NFκB p65 as well as the pro-inflammatory cytokines IL-1β, IL-6, and TNFα. This study thus shows that attenuation of the severity of TLR2- and TLR4-mediated post-stroke inflammation ameliorates ischemic brain damage.