Neeraj K. Agrawal

Atherosclerosis in diabetes mellitus: role of inflammation.

Inflammation plays a central role in the pathophysiology of atherosclerosis, starting from initiation, through progression and ultimately the thrombotic complications of atherosclerosis. Diabetes mellitus is a major risk factor for atherosclerosis. Hyperglycemia-induced endothelial dysfunctions, along with hypercoagulable potential of diabetes mellitus, accelerate the process of atherothrombotic complications. Therefore, clinically feasible markers to monitor subtle systemic inflammatory burden and specific add-on therapy for the same constitute need of the present day. The understanding of the concept of inflammation in diabetes-accelerated atherosclerosis can be used practically to predict future cardiovascular risk by evaluating inflammatory biomarkers and to design clinical trials making inflammation as a therapeutic target.

Association of Toll-like receptor 4 polymorphisms with diabetic foot ulcers and application of artificial neural network in DFU risk assessment in type 2 diabetes patients.

The Toll-Like receptor 4 (TLR4) plays an important role in immunity, tissue repair, and regeneration. The objective of the present work was to evaluate the association of TLR4 single nucleotide polymorphisms (SNPs) rs4986790, rs4986791, rs11536858 (merged into rs10759931), rs1927911, and rs1927914 with increased diabetic foot ulcer (DFU) risk in patients with type 2 diabetes mellitus (T2DM). PCR-RFLP was used for genotyping TLR4 SNPs in 125 T2DM patients with DFU and 130 controls. The haplotypes and linkage disequilibrium between the SNPs were determined using Haploview software. Multivariate linear regression (MLR) and artificial neural network (ANN) modeling was done to observe their predictability for the risk of DFU in T2DM patients. Risk genotypes of all SNPs except rs1927914 were significantly associated with DFU. Haplotype ACATC (P value = 9.3E − 5) showed strong association with DFU risk. Two haplotypes ATATC (P value = 0.0119) and ATGTT (P value = 0.0087) were found to be protective against DFU. In conclusion TLR4 SNPs and their haplotypes may increase the risk of impairment of wound healing in T2DM patients. ANN model (83%) is found to be better than the MLR model (76%) and can be used as a tool for the DFU risk assessment in T2DM patients.

A Functional Single Nucleotide Polymorphism -1562C>T in the Matrix Metalloproteinase-9 Promoter Is Associated With Type 2 Diabetes and Diabetic Foot Ulcers

Impaired neovascularization is the hallmark of type 2 diabetes, which results in various macro- and microvascular complications and the development of foot ulcerations later in life. Matrix metalloproteinases (MMPs) are the key enzymes which influence matrix remodeling. Here, we aim to investigate that whether single nucleotide polymorphism (SNP -1562C>T) (rs3918242) in the promoter region of MMP-9 gene, which alters the transcriptional activity of MMP-9 is associated with type 2 diabetes and diabetic foot ulcers (DFUs). This case–control study was composed of 730 individuals, out of which 463 patients were with type 2 diabetes mellitus (T2DM) and 267 were nondiabetic healthy controls (non-DM controls). T2DM patients were subclassified as 149 cases without any secondary complications (T2DMNSC), 110 with DFUs, 204 T2DM patients having one or the other secondary complications. Genotyping for -1562C>T SNP in MMP-9 gene was done by polymerase chain reaction–restriction fragment length polymorphism method and sequencing. SNP -1562C>T of MMP-9 gene showed a significant association with T2DM and DFU. The allele distribution differed significantly between patients and normal control group (odds ratio = 1.82, P = .00005, 95% confidence interval = 1.36-2.42 for T2DM vs control and odds ratio = 2.112, P = .00048, 95% confidence interval = 1.38-3.126 for DFU vs control) indicating strong association of SNP -1562C>T of MMP-9 gene with T2DM and DFU in an Indian population. SNP -1562C>T in the promoter of the MMP-9 gene results in increased expression at the level of the transcription. To the best of our knowledge, this is the first report that suggests that SNP -1562C>T in the promoter of the MMP-9 gene is associated with T2DM and DFU. An increased MMP-9 production from high expressing T allele may promote matrix degradation.

Combined Effect of GSTT1 and GSTM1 Polymorphisms on Human Male Infertility in North Indian Population

Genes of different pathways regulate spermatogenesis, and complexity of spermatogenic process indicates that polymorphisms or mutations in these genes could cause male infertility. Detoxification pathway is involved in the regulation of spermatogenesis by reducing oxidative stress and contributes to the maintenance of global methylation in concert with other pathways. Glutathione S-transferases (GSTs) belong to the family of phase II antioxidant enzymes involved in the cellular detoxification of various physiological substances. Glutathione S-transferases act as an antioxidant and protect spermatozoa from oxidative stress. Increase in the levels of reactive oxygen species (ROS) along with reduced activity of GSTs may result in sperm membrane damage and DNA fragmentation. A case–control study was done to elucidate the role of deletion polymorphism of GSTT1 and GSTM1 genes from GSTs family on idiopathic human male infertility. The study comprises 2 groups: 113 nonobstructive azoospermia patients and 91 healthy fertile controls. Genomic DNA was analyzed by polymerase chain reaction for GSTT1 and GSTM1 genes. The study showed statistically significant protective association of GSTT1 null genotype with human male infertility (odds ratio [OR]: 0.3, 95% confidence interval [CI] 0.143-0.9966, P = .048) but not with GSTM1 null genotype (OR: 0.66, 95% CI 0.3653-1.2234, P = .19). Also, combination of null genotypes of GSTM1 and GSTT1 confers protective effect (OR: 0.28, CI 0.0801-0.948; P = .04). Probably, individuals bearing GSTM1 and GSTT1 (−/−) genotypes may have protective effect by gene–gene interaction mechanism. In summary, our study underscores the significance of combined effect of GSTT1 and GSTM1 null genotypes in modulating the risk of male infertility.

Increased expression of TLR9 associated with pro-inflammatory S100A8 and IL-8 in diabetic wounds could lead to unresolved inflammation in type 2 diabetes mellitus (T2DM) cases with impaired wound healing

BACKGROUND Type 2 diabetes mellitus (T2DM) is characterized by persistent hyperglycemia which causes a chain of abrupt biochemical and physiological changes. Immune dys-regulation is the hallmark of T2DM that could contribute to prolonged inflammation causing transformation of wounds into non-healing chronic ulcers. Toll like receptor -9 (TLR9) is a major receptor involved in innate immune regulation. TLR9 activation induces release of pro-inflammatory molecules like S100A8 and interleukin-8 (IL-8) by myeloid cells causing migration of myeloid cells to the site of inflammation. We hypothesized that pro-inflammatory S100A8 and IL-8 proteins could cause persistent inflammation in chronic wounds like diabetic foot ulcer (DFU) and may contribute to impaired wound healing in T2DM patients. MATERIALS AND METHODS Expression of TLR9 and its downstream effector molecules S100A8, and IL-8 were analyzed in chronic diabetic wound and non-diabetic control wound tissue samples by semiquantitative reverse transcriptase - polymerase chain reaction (RT-PCR), quantitative RT-PCR, western blot and immunofluorescence. CD11b(+)CD33(+) myeloid cells were analyzed by flow cytometry. RESULTS TLR9 message and protein were higher in diabetic wounds compared to control wounds (p=0.03, t=2.21 for TLR9 mRNA; p=<0.001, t=4.21 for TLR9 protein). TLR9 down-stream effector molecules S100A8 and IL-8 were also increased in diabetic wounds (p=0.003, t=3.1 for S100A8 mRNA; p=0.04, t=2.04 for IL-8). CD11b(+) CD33(+) myeloid cells were decreased in T2DM as compared to non-diabetic controls (p=0.001, t=3.6). DFU subjects had higher levels of CD11b(+) CD33(+) myeloid cells as compared to non-DFU T2DM control (p=0.003, t=2.8). Infection in the wound microenvironment could be the cause of increase in CD11b(+)CD33(+) myeloid cells in DFU (p=0.03, t=2.5). CONCLUSION The up-regulation of myeloid cell-derived pro-inflammatory molecules S100A8 and IL-8 in combination with lower levels of CD11b(+) CD33(+) myeloid cells may cause the impairment of wound healing in T2DM subjects leading to chronic ulcers.

Association of Variant rs7903146 (C/T) Single Nucleotide Polymorphism of TCF7L2 Gene With Impairment in Wound Healing Among North Indian Type 2 Diabetes Population A Case–Control Study

The variants of transcription factor 7-like 2 (TCF7L2) gene have been shown to be associated with type 2 diabetes mellitus (T2DM) and its several secondary complications. Here, we aimed to examine the possible role of one of the common variant of this gene, rs7903146 (C/T), with impairment of wound healing in cases with T2DM. A total of 750 individuals, including 322 patients with T2DM and 120 patients with diabetic foot ulcers (DFUs) and 308 controls, were analyzed for rs7903146 variant of the TCF7L2 gene. Genotyping was done by polymerase chain reaction–restriction fragment length polymorphism. For rs7903146 variant, TT genotype frequency in patients with DFU was 10.8% and in controls was 5.2%. Risk genotype (TT) frequencies showed statistically significant difference between the DFU patients versus non-DM control group (odds ratio = 2.44, P = .037, 95% confidence interval = 1.05-5.64) compared with nonrisk genotype (CC + CT). In the present study, a positive significant association between DFU and the TT genotype of rs7903146 (C/T) variant of TCF7L2 gene was found.

CT angiographic evaluation of perforators in the lower limb and their reconstructive implication

Background: The perforator flaps evolved on the knowledge of the vascular tree from the main vascular trunk up to the subdermal plexus. Therefore, we thought that its necessary to map the whole vascular arcade by CT angiography. The aim of this study is to evaluate the perforators and the whole vascular tree of the lower limb by peripheral CT angiography with 3D reconstruction and intraoperative evaluation. This study helps in designing flaps of different constituents based on the selected perforators. Materials and Methods: Twenty patients having lower limb defects were selected. CT angiography was done using a non-ionic iodinated contrast media injected through the antecubital vein. The lower limbs were imaged using volume rendering CT scan machine. Three dimensional reconstructions were made. The whole arterial tree, along with the perforators, were mapped. Findings of the audio-Doppler were correlated with the CT angiographic observations. Further these evaluations were confirmed by intraoperative findings. Results: The three dimensional CT angiographic reconstruction with bone and soft tissue provided advanced knowledge of this vascular network. It delineated the main vessel, the perforators, their caliber, distance from fixed bony landmarks and course up to the subdermal plexus. These findings were confirmed during dissection of the proposed flap. The perforators were mainly musculocutaneous in the proximal leg and septocutaneous distally. Conclusions: The vascular details visualized by this technique made advancement over the existing methods namely color Doppler, audio Doppler, two dimensional angiography etc. It improved the understanding of perforator flaps and their successful clinical application.

Decreased expression of heat shock proteins may lead to compromised wound healing in type 2 diabetes mellitus patients

BACKGROUND Heat shock proteins (HSPs) are inducible stress proteins expressed in cells exposed to stress. HSPs promote wound healing by recruitment of dermal fibroblasts to the site of injury and bring about protein homeostasis. Diabetic wounds are hard to heal and inadequate HSPs may be important contributors in the etiology of diabetic foot ulcers (DFU). OBJECTIVE To analyze the differential expression of HSPs and their downstream molecules in human diabetic wounds compared to control wounds. METHODS Expressional levels of HSP27, HSP47 and HSP70 and their downstream molecules like TLR4, p38-MAPK were seen in biopsies from 101 human diabetic wounds compared to 8 control subjects without diabetes using RT-PCR, western blot and immunohistochemistry. RESULTS Our study suggested a significant down regulation of HSP70, HSP47 and HSP27 (p value=<0.001 for HSP70; p value=0.007 for HSP47; p value=0.007 for HSP27) in DFU along with their downstream molecules TLR4 and p38-MAPK (p value=0.006 for p38-MAPK; p value=0.02 for TLR4). HSP70 levels were significantly lower in male subjects and their levels increased significantly with the grades of wound on Wagners scale. Infection status of the wounds was found to be significantly associated with the increased levels of HSP70 and HSP27 in infected diabetic wounds. CONCLUSIONS Our study demonstrates that the down regulation of HSPs in diabetic wounds is associated with wound healing impairment in T2DM subjects.

Genetic and epigenetic alterations in Toll like receptor 2 and wound healing impairment in type 2 diabetes patients

AIM Persistent hyperglycemic microenvironment in type 2 diabetes mellitus (T2DM) leads to the development of secondary complications like wound healing impairment. Proper co-ordination of innate immune system plays an integral role in wound healing. Toll like receptors (TLRs) are prominent contributors for the induction of the innate immune and inflammation response. TLR2 is an important extracellular member in mammalian TLR family and has been shown to be a potent player in the wound healing mechanism. METHODS Expressional status of TLR2 was seen in wounds of T2DM cases with respect to the severity of wounds in 110 human lower extremity wounds. The methylation status of TLR2 promoter was also examined. RESULTS Although TLR2 transcripts were downregulated in T2DM wounds compared to control, their levels tend to increase with the severity of T2DM wounds. The methylation status of TLR2 gene promoter was not significantly different among different grades of wounds in T2DM subjects. The CpG sites investigated were totally or partially methylated in majority of DFU cases. CONCLUSION TLR2 down regulation in wounds of T2DM patients compared to non diabetic patients may lead to development of non healing chronic ulcers in them.

Dysregulation of apoptotic pathway candidate genes and proteins in infertile azoospermia patients.

OBJECTIVE To dissect the role of the apoptotic pathway and its regulation in the pathogenesis of male infertility in nonobstructive azoospermia. DESIGN Prospective study. SETTING University hospital. PATIENT(S) Sixty-three infertile azoospermic patients with different histologic phenotypes were recruited (obstructive azoospermia, n = 16; hypospermatogenesis, n = 11; maturation arrest, n = 15; Sertoli cell only, n = 21). INTERVENTION(S) Testicular biopsies for histopathologic and expression analysis. MAIN OUTCOME MEASURE(S) Expression analysis by quantitative reverse transcription-polymerase chain reaction, protein localization by immunohistochemistry and apoptotic proteome array. RESULT(S) Results showed significantly increased expression of proapoptotic proteins like BAX, BAD, and BAK and comparatively lowered expression of antiapoptotic BCL2 and BCLW. Immunostaining revealed increased active caspase-3 activity and more TUNEL-positive cells in different impaired phenotypes as compared with normal. In addition, significantly increased m-RNA expression of TGFB1, P53, and FASLG along with significant down-regulation of VEGFA were observed. Expression of phosphorylated P53 at the S15 position and phosphorylated RAD17 at S635 was observed in cases with spermatogenic impairment at the translational level. CONCLUSION(S) The results clearly indicate increased levels of apoptosis along with its other regulatory factors. The balance between pro- (BAX and BAK) and antiapoptotic (BCL2 and BCLW) genes was disturbed, which may lead to altered apoptosis. Therefore, altered regulation of apoptosis might be associated with impaired spermatogenesis, eventually leading to male infertility.