Qamar Javed

Galectin 1 is involved in vascular smooth muscle cell proliferation

OBJECTIVE Smooth muscle cell (SMC) migration and proliferation are the key steps in the development of atherosclerosis and restenosis. Matricellular proteins have been implicated in cell adhesion, migration and proliferation. Here we investigated the role of the matricellular protein galectin-1 (Gal-1), a beta-galactoside-binding lectin, in SMC proliferation in atheroma and DNA synthesis in cell culture. METHODS Protein expression was visualised by tissue section immunostaining. RNA expression was analysed using Northern blot analysis. DNA synthesis of human vascular SMCs was determined by 3H-thymidine incorporation. Recombinant glutathione S-transferase-galectin-1 fusion protein (Gal FP) binding to extracellular matrix (ECM) proteins was measured by ELISA. Gal-1 binding to cells and ECM was estimated using 125I-labelled Gal FP. RESULTS Prominent Gal-1 staining coincided with SMC proliferation in human coronary endarterectomy samples in organoid culture. In cell culture, Gal-1 mRNA was upregulated in growing SMCs. Gal FP increased serum-induced DNA synthesis of human SMCs on plastic or endogenous ECM, but not of a rat PAC1 SM cell line. Also, Gal FP slightly increased SMC adhesion to ECM. SMCs exhibited a complex pattern of receptor-ligand interactions with Gal FP. The Gal-1 binding to SMCs was much stronger than to ECM, produced by these SMCs. We identified new ECM proteins: thrombospondin, vitronectin and osteopontin, which bound to Gal FP in a dose- and beta-galactoside-dependent manner in ELISA. CONCLUSIONS Gal-1 binding to SMCs was stronger than to ECM, although ECM of atherosclerotic blood vessels contained additional ECM proteins which bound to Gal-1. Gal-1 was upregulated during SMC growth and Gal FP enhanced serum-induced DNA synthesis in SMCs. Overall, Gal-1 upregulation is likely to provide a reinforcement of serum-induced events during vascular injury.

Loss-of-function mutations of ILDR1 cause autosomal-recessive hearing impairment DFNB42.

By using homozygosity mapping in a consanguineous Pakistani family, we detected linkage of nonsyndromic hearing loss to a 7.6 Mb region on chromosome 3q13.31-q21.1 within the previously reported DFNB42 locus. Subsequent candidate gene sequencing identified a homozygous nonsense mutation (c.1135G>T [p.Glu379X]) in ILDR1 as the cause of hearing impairment. By analyzing additional consanguineous families with homozygosity at this locus, we detected ILDR1 mutations in the affected individuals of 10 more families from Pakistan and Iran. The identified ILDR1 variants include missense, nonsense, frameshift, and splice-site mutations as well as a start codon mutation in the family that originally defined the DFNB42 locus. ILDR1 encodes the evolutionarily conserved immunoglobulin-like domain containing receptor 1, a putative transmembrane receptor of unknown function. In situ hybridization detected expression of Ildr1, the murine ortholog, early in development in the vestibule and in hair cells and supporting cells of the cochlea. Expression in hair cell- and supporting cell-containing neurosensory organs is conserved in the zebrafish, in which the ildr1 ortholog is prominently expressed in the developing ear and neuromasts of the lateral line. These data identify loss-of-function mutations of ILDR1, a gene with a conserved expression pattern pointing to a conserved function in hearing in vertebrates, as underlying nonsyndromic prelingual sensorineural hearing impairment.

Molecular maturation of cell adhesion systems during mouse early development

During cleavage, the mouse embryo expresses a variety of cell adhesion systems on its cell surfaces. We have reviewed biogenetic and assembly criteria for the formation of the uvomorulin/catenin, tight junction and desmosome adhesion systems as the trophectoderm differentiates. Each system reveals different mechanisms regulating molecular maturation. Adhesion processes contribute to the generation of distinct tissues in the blastocyst by modifying the expression pattern of blastomeres entering the non-epithelial inner cell mass lineage. Cell adhesion also influences the spatial organisation, but rarely the timing of expression, of proteins involved in trophectoderm differentiation.

Biogenesis of structural intercellular junctions during cleavage in the mouse embryo

SUMMARY The preimplantation embryo differentiates the trophectoderm epithelium which, from the 32-cell stage, generates the blastocoel of the blastocyst and, after implantation, gives rise to most extraembryonic lineages of the conceptus. Trophectoderm differentiation begins at compaction (8-cell stage) when cell-cell adhesion, mediated by uvomorulin, and epithelial cell polarisation first occur. Here, we review our work on the biogenesis of tight junctions and desmosomes during epithelial differentiation. Tight junction construction begins at compaction and appears to be a gradual process, both at morphological and molecular levels. This maturation pattern may be due in part to sequential expression of tight junction constituents from the embryonic genome. Tight junction formation is dependent upon uvomorulin adhesion but can be inhibited by different means without apparently disturbing cell adhesion or polarisation. Cell interactions appear to regulate tight junction tissue specificity, in part by controlling the level of synthesis of constituents. Desmosome formation begins at the 32-cell stage, particularly as the embryo initiates blastocoel accumulation, and, in contrast with tight junction formation, does not appear to be a gradual process. Thus, nascent desmosomes appear mature in terms of their molecular composition. Desmosomal proteins are synthesised well in advance of desmosome formation but the synthesis of the principal glycoprotein components begins at the blastocyst stage and may regulate the timing of junction assembly. Implications of these differing patterns of biogenesis for the embryo are discussed.

Therapeutic Potential of Tumour Necrosis Factor-alpha Antagonists in Patients with Chronic Heart Failure

OBJECTIVES Elevated levels of tumour necrosis factor alpha (TNF-alpha) are associated with the development of heart failure. TNF-alpha antagonists, etanercept (ETA) and infliximab (INF) have been used for treating different clinical conditions. Anti-TNF-alpha therapy did not show favourable results in patients with chronic heart failure (CHF). This review analyses the reasons for anti-TNF-alpha therapy failure in CHF patients; potential approaches for improved clinical outcome are also addressed. DESIGN AND METHODS Data analysis from clinical trials of CHF patients treated with ETA and INF. RESULTS In heart failure patients, ETA and INF therapy did not lead to improved clinical outcomes and high doses of INF were associated with worsening of cardiac events. This contrasts with the observation that these agents are associated with reduced cardiac events when used in patients with inflammatory diseases such as rheumatoid arthritis (RA). CONCLUSIONS Treatment of CHF patients with TNF-alpha antagonists did not show encouraging results. There is a need for the development of better treatment strategies for CHF. Perhaps, tailored anti-TNF-alpha therapy in relation to the TNF-alpha genotype of CHF patients and targeting of the cytokine gene expression via signalling pathway inhibitors may have useful clinical implications.

In vitro evaluation of vascular endothelial growth factor (VEGF)-eluting stents

BACKGROUND Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen. It promotes re-endothelialisation of the damaged vessel surface seen after stenting. Stent thrombosis and in-stent restenosis are partly related to endothelial denudation caused by stent implantation. We propose using hydrocarbon polymer-coated stents immersed in VEGF to speed re-endothelialisation and reduce the risk of stent thrombosis and restenosis. METHODS Stents (3 x 20 mm) were immersed in VEGF solutions and maximal VEGF absorption calculated. VEGF release from these stents was measured in a perfusion circuit. Delivery of VEGF to arterial wall was measured. Sterile VEGF-loaded stents were cultured with human umbilical vein endothelial cells. RESULTS 18.5 +/- 4.1 microg VEGF was absorbed, 80% of which was released over 9 days; 11 +/- 6.8% of the initial VEGF loaded was delivered to the vessel wall. Cells exposed to VEGF-eluting stents showed an 11% increase in growth relative to controls. CONCLUSION VEGF may be a candidate for stent-based delivery and may increase the rate of endothelialisation in vivo.

Association of Interleukin-6 Gene Promoter Polymorphism with Coronary Artery Disease in Pakistani Families

Interleukin-6 (IL-6) is a well-known inflammatory cytokine and suggested to be involved in the development of coronary artery disease (CAD). IL-6 gene expression has been investigated with controversy in CAD patients. This study investigates the association of the IL-6 gene expression with CAD, the molecular basis for the regulation of interleukin-6 expression in a Pakistani population. Our data show that the serum IL-6 levels were increased in patients with CAD compared with healthy controls and that the IL-6 gene polymorphism at -174 was more prevalent in CAD cases. There was a statistically significant association between the IL-6 gene polymorphism and CAD, which may be associated with an increased risk for the disease. Moreover, circulating IL-6 and hs-CRP levels were significantly higher in patients with CC genotype (P < 0.0001 and P < 0.0001, resp.). In a binary logistic-regression model, an independent association was found between CAD and increased serum IL-6 and hs-CRP levels and -174G>C polymorphism. This is the first report on the IL-6 expression and the IL-6 gene polymorphism in patients with CAD from Pakistan, and hence it highlights a novel risk factor for the disease.

Resistin gene promoter region polymorphism and the risk of hypertrophic cardiomyopathy in patients

Resistin, a novel cytokine, is associated with an inflammatory process and is suggested to induce hypertrophy in rat cardiomyocytes. Resistin gene expression has not been investigated in patients with hypertrophic cardiomyopathy (HCM). This study investigates resistin levels in HCM patients and healthy controls and the molecular basis for the regulation of the resistin gene (RETN) in a Pakistani population. Patients with HCM (n = 105) and healthy individuals (n = 110) were enrolled in this investigation. Serum resistin levels were determined by enzyme-linked immunosorbent assay (ELISA). RETN genotyping was performed by polymerase chain reaction (PCR) and DNA sequencing. Our data showed a statistically significant increase in resistin levels from HCM patients compared with healthy subjects (6.3 +/- 2.7 ng/mL in patients vs 3.4 +/- 2.1 ng/mL in controls, P < 0.0001). The RETN -420 C > G polymorphism was significantly high in patients with HCM compared with the control group (P < 0.001). There was a significant difference between the C and G alleles from HCM cases and controls (odds ratio [OR] = 3.54, 95% confidence interval [CI] = 2.36-5.30, P < 0.0001). Logistic-regression analysis showed that the increased resistin levels, and the RETN-420 C > G polymorphism were significantly associated with HCM. Our data suggest that the elevated resistin levels and the RETN -420 C > G polymorphism may be associated with cardiac hypertrophy in the study population.

Tumor Necrosis Factor-Alpha Gene Promoter Region Polymorphism and the Risk of Coronary Heart Disease

Background. Tumor necrosis factor-alpha (TNF-α) gene polymorphisms have been implicated in the manifestation of atherosclerosis. Controversy exists regarding the link between the cytokines variant genotype and CHD among different ethnic groups. There have been fewer studies on the TNF-α gene −1031T>C and −863C>A polymorphisms in relation to CHD. Therefore, the current study was designed to investigate the association of the TNF-α gene −1031T>C and −863C>A polymorphisms with CHD in a Pakistani population. Methods. Patients with CHD (n = 310) and healthy individuals (n = 310) were enrolled in this study. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results. A significant difference was observed in the −863C>A polymorphism between patients with CHD and control subjects (P < 0.0001). CHD risk was positively associated with the variant allele −863A (P < 0.0001) in the study subjects. There was no significant link between the −1031T>C polymorphism and CHD risk in the study population. Haplotypes A-T and A-C of the TNF-alpha gene loci at −863 and −1031 showed higher frequency in the patient group compared with controls (P < 0.05). Conclusion. The TNF-α  −863C>A gene polymorphism was associated with the pathogenesis of CHD while the −1031T>C polymorphism did not show any link with the disease in a Pakistani population.

Heritability of genetic variants of resistin gene in patients with coronary artery disease: a family-based study.

OBJECTIVE The resistin gene (RETN) -420C>G and +299G>A polymorphism was investigated in a case-control study from forty complex Pakistani families with coronary artery disease (CAD) history. Heritability of the susceptible/variant alleles was investigated from parent-offspring trios in these families. METHOD Resistin levels were determined from 239 individuals by enzyme-linked immunosorbent assay. Genotyping was performed using polymerase chain reaction and restriction fragment length polymorphism. RESULTS Elevated resistin levels were observed from CAD cases vs. controls (p<0.0001). The RETN -420C>G and +299G>A polymorphism was more prevalent in cases vs. controls (p<0.0001). The transmission disequilibrium test revealed a significant association of the -420 and +299 polymorphism with CAD (χ(2)=34.4, p<0.0001 and χ(2)=31.6, p<0.0001, respectively). CONCLUSION Elevated resistin, and the RETN -420C>G and +299G>A polymorphism may contribute to familial CAD. The -420 and +299 variant alleles are transmitted more frequently from parent to affected offspring. This is the first report on the association of the RETN +299G>A polymorphism with CAD.