Ramareddy V. Guntaka

Regulation of collagen degradation in the rat myocardium after infarction

Fibrillar collagens, essential for maintaining the structural integrity of the myocardium, are degraded by matrix metalloproteinase (MMP-1). In other tissues collagenolysis is an important component of wound healing. Here we examined collagen degradation in the myocardium after infarction. Collagenase activity, measured by zymography, and expression of matrix metalloproteinase (MMP-1) and tissue inhibitor of metalloproteinase (TIMP) mRNA, detected by Northern blotting and in situ hybridization, in the rat heart 6 h to 28 days after left coronary artery ligation were studied. Sham-operated rats served as controls. Infarcted left ventricle was compared to non-infarcted right ventricle and interventricular septum and to sham-operated tissues. We found a transient increase in collagenase activity in the infarcted left ventricle, which began at day 2 (4.5-fold increase compared to controls), peaked at day seven (6.5-fold increase) and declined thereafter, together with a concomitant increase and contribution in collagenolytic activity of gelatinases (MMP-2 and MMP-9). An increase in collagenase mRNA was not seen until day 7 and only in the infarcted ventricle, while changes in MMP-1 activity or mRNA expression were not observed at remote sites or in sham-operated controls. Transcription of TIMP mRNA was observed at 6 h (two-fold increase) in the infarcted ventricle, peaked on day two after MI (eight-fold increase) and slowly decreased thereafter. No change in TIMP mRNA expression was observed at remote sites or in sham-operated controls. Cells responsible for transcription of MMP-1 and TIMP mRNA were fibroblast-like cells, not inflammatory or endothelial cells. At the site of infarction post-translational activation of latent collagenase (MMP-1) plays a greater role in the wound healing response than transcription of collagenase mRNA. Collagenase mRNA is synthesized when the latent extracellular pool of MMP-1 is reduced through the activation of latent collagenases and gelatinases. TIMP mRNA synthesis is regulated by the activation of MMPs with the balance between collagenase activation and TIMP inhibition determining the amount of collagenolysis in infarcted tissue.

Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA.

Antisense oligodeoxynucleotides, triplex-forming oligodeoxynucleotides and double-stranded small interfering RNAs have great potential for the treatment of many severe and debilitating diseases. Concerted efforts from both industry and academia have made significant progress in turning these nucleic acid drugs into therapeutics, and there is already one FDA-approved antisense drug in the clinic. Despite the success of one product and several other ongoing clinical trials, challenges still exist in their stability, cellular uptake, disposition, site-specific delivery and therapeutic efficacy. The principles, strategies and delivery consideration of these nucleic acids are reviewed. Furthermore, the ways to overcome the biological barriers are also discussed so that therapeutic concentrations at their target sites can be maintained for a desired period.

Role of single-stranded DNA regions and Y-box proteins in transcriptional regulation of viral and cellular genes

Single‐stranded regions, known to be important for optimal rates of transcription, have been observed in the promoters of several cellular genes as well as in the promoters of many pathogenic viruses. Several host‐encoded, single‐stranded DNA binding proteins capable of binding these regions have been purified and their genes isolated. In this review, information available about single‐stranded regions present within various promoters and the interaction of a novel class of single‐stranded DNA binding transcription factors belonging to the Y‐box family of proteins is reviewed. Mechanisms by which these proteins influence transcription of both cellular and viral genes are proposed.

Triplex-forming oligonucleotides as modulators of gene expression

Triplex-forming oligonucleotides (TFOs) have gained prominence in the recent years because of their potential applications in antigene therapy. In particular they have been used as (i) inducers of site-specific mutations, (ii) reagents that selectively and specifically cleave target DNA, and (iii) as modulators of gene expression. In this mini-review, we have made an attempt to highlight the characteristics of these TFOs and the effects of various modifications in the phosphate backbone as well as in the purine and pyrimidine moieties, which contribute to the stability and efficiency of triplex formation. Studies to explore the mechanism of down-regulation of transcription of various genes suggest that at least some TFOs exert their effect by inhibiting binding of specific transcription factors to their cognate cis-acting elements. Recent reports indicate the presence of these potential triplex-forming DNA structures in the genomes of prokaryotes and eukaryotes that may play a major role in target site selection and chromosome segregation as well as in the cause of heritable diseases. Finally, some potential problems in the development of these TFOs as antigene therapeutic agents have also been discussed.

Angiotensin II and extracellular matrix homeostasis

As a circulating hormone, endocrine properties of angiotensin (Ang) II are integral to circulatory homeostasis. Produced de novo its autocrine/paracrine properties contribute to biologic responses involving various connective tissues (e.g. extracellular matrix, adipose tissue, bone and its marrow). In this brief review, we develop the concept of extracellular matrix homeostasis, a self regulation of cellular composition and structure, wherein fibroblast-derived AngII regulates elaboration of TGF-beta 1, a fibrogenic cytokine responsible for connective tissue formation at normal and pathologic sites of collagen turnover.

Evidence for extensive methylation of ribosomal RNA genes in a rat XC cell line

XC cell line was established from a rat fibrosarcoma induced by avian sarcoma virus. Analysis of ribosomal RNA genes in the DNA of this cell line indicated that there was a 3- to 4-fold increase in the number of the rDNA repeating units. [methyl-14C]Methionine labeling experiments as well as Southern blot analysis of the restriction endonuclease fragments showed extensive methylation of the cytosine residues in the rDNA. Further analysis by HpaII, HhaI and MspI suggested that the entire rDNA repeat unit was uniformly methylated in the recognition sequence for these enzymes. Parallel experiments with two other rat cell lines (rat embryo fibroblasts and adenovirus-transformed rat embryo cells) indicated no evidence of methylation in the rDNA.

Enhanced Hepatic Uptake and Bioactivity of Type α1(I) Collagen Gene Promoter-Specific Triplex-Forming Oligonucleotides after Conjugation with Cholesterol

A triplex-forming oligonucleotide (TFO) specific for type α1(I) collagen promoter is a promising candidate for treating liver fibrosis. Earlier, we determined the pharmacokinetics and biodistribution of TFO after systemic administration into normal and fibrotic rats. In this study, we conjugated cholesterol to the 3′ end of the TFO via a disulfide bond and determined its cellular and nuclear uptake and bioactivity using HSC-T6 cell lines in vitro, followed by biodistribution at whole-body, organ (liver), and subcellular levels. Conjugation with cholesterol had little effect on the triplex-forming ability of the TFO with target duplex DNA, and the cellular uptake of 33P-TFO-cholesterol (Chol) increased by 2- to approximately 4-fold. Real-time reverse transcriptase-polymerase chain reaction analysis after transfection of HSC-T6 cells with TFO-Chol or TFO indicated that TFO-Chol had higher inhibition on type α1(I) collagen primary transcript than naked TFO at low concentration (200 nM) but showed similar inhibition at higher concentration (500 and 1000 nM). There was increase in the inhibition on primary transcript with transfection time. The hepatic uptake of 33P-TFO-Chol after systemic administration was 72.22% of the dose compared with 45.8% of 33P-TFO. There was significant increase in the uptake of 33P-TFO-Chol by hepatic stellate cells and hepatocytes. More importantly, the nuclear uptake of TFO-Chol was higher than TFO in cell culture system and in vivo studies. In conclusion, TFO-Chol is a potential antifibrotic agent.

TRIPLE HELIX-FORMING OLIGONUCLEOTIDE CORRESPONDING TO THE POLYPYRIMIDINE SEQUENCE IN THE RAT ALPHA 1(I) COLLAGEN PROMOTER SPECIFICALLY INHIBITS FACTOR BINDING AND TRANSCRIPTION

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of α1(I) collagen promoter between nucleotides −200 and −140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between −172 and −138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of α1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.

Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets

Islet transplantation is limited by islet graft failure due to poor revascularization, host immune rejection and nonspecific inflammatory response. Delivery of human vascular endothelial growth factor (hVEGF) gene to the islets is likely to promote islet revascularization and survival. We used a bicistronic adenoviral vector encoding hVEGF and CpG-free allele of green fluorescent protein (Adv-GFP-hVEGF) and introduced into human pancreatic islets by transfection. We found that transfection efficiency and apoptosis were dependent on the multiplicity of infection (MOI). Compared to Adv-GFP transfected and nontransfected islets, the levels of hVEGF secreted from Adv-GFP-hVEGF transfected islets were higher and exhibit a linear relationship between hVEGF expression and MOI (10–5000). Persistent, but low level expression of hVEGF from nontransfected islets was also observed. This may be due to expression of the endogenous hVEGF gene under hypoxic conditions. The levels of DNA fragmentation determined by ELISA of islet lysates were dependent on the MOI of Adv-GFP-hVEGF. On glucose challenge, insulin release from transfected islets was comparable to nontransfected islets. Immunohistochemical staining for hVEGF was very high in Adv-GFP-hVEGF transfected islets. Weak staining was also observed for hCD31 in both transfected and nontransfected islets. These findings suggest that Adv-GFP-hVEGF is a potential candidate for promoting islet revascularization.

Aldosteronism in heart failure: a proinflammatory/fibrogenic cardiac phenotype. Search for biomarkers and potential drug targets.

Heart failure is a major health problem of epidemic proportions. Irrespective of its etiologic origins, a dysfunction of this normally efficient muscular pump is associated with systemic consequences, a progressive downhill clinical course and poor prognosis. Ventricular dysfunction is ultimately accompanied by neurohormonal system activation that accounts for: the congestive heart failure syndrome; an induction of oxi/nitrosative stress; adverse vascular remodeling; and activation of the immune system that contributes to a wasting syndrome known as cardiac cachexia. Circulating effector hormones of the renin-angiotensin-aldosterone system are an integral feature of this neurohormonal activation; they have systemic consequences. Insights into the pathophysiology of heart failure will identify improved methods of prevention, including biomarkers to aid in its detection and identification of risk, and to the development of specific drug targets. Herein we address one aspect of the neurohormonal profile of heart failure, namely that related to aldosteronism. Our focus is directed at the link between aldosteronism and its adverse influence on coronary vasculature structure, a proinflammatory/fibrogenic cardiac phenotype, which is based on an immunostimulatory state that includes activated peripheral blood mononuclear cells.