Regression of Established Cardiac Fibrosis in Hypertensive Heart Disease.

Established cardiac fibrosis (ECF) with symptomatic heart failure preserved ejection fraction represents an ever-increasing segment of the hypertensive population. The regression of ECF with attendant improvement in myocardial stiffness and symptomatic failure represents an unmet health care need. Is the regression of ECF in hypertensive heart disease feasible and will stiffness and symptomatic failure be improved? What is the cellular/molecular signaling involved in its regression? What incremental knowledge is needed to proceed effectively? These issues are addressed in this Review.

Myofibroblast secretome and its auto-/paracrine signaling.

Myofibroblasts (myoFb) are phenotypically transformed, contractile fibroblast-like cells expressing α-smooth muscle actin microfilaments. They are integral to collagen fibrillogenesis with scar tissue formation at sites of repair irrespective of the etiologic origins of injury or tissue involved. MyoFb can persist long after healing is complete, where their ongoing turnover of collagen accounts for a progressive structural remodeling of an organ (a.k.a. fibrosis, sclerosis or cirrhosis). Such persistent metabolic activity is derived from a secretome consisting of requisite components in the de novo generation of angiotensin (Ang) II. Autocrine and paracrine signaling induced by tissue AngII is expressed via AT1 receptor ligand binding to respectively promote: i) regulation of myoFb collagen synthesis via the fibrogenic cytokine TGF-ß1-Smad pathway; and ii) dedifferentiation and protein degradation of atrophic myocytes immobilized and ensnared by fibrillar collagen at sites of scarring. Several cardioprotective strategies in the prevention of fibrosis and involving myofibroblasts are considered. They include: inducing myoFb apoptosis through inactivation of antiapoptotic proteins; AT1 receptor antagonist to interfere with auto-/paracrine myoFb signaling or to induce counterregulatory expression of ACE2; and attacking the AngII-AT1R-TGF-ß1-Smad pathway by antibody or the use of triplex-forming oligonucleotides.

Zinc and the prooxidant heart failure phenotype.

Neurohormonal activation with attendant aldosteronism contributes to the clinical appearance of congestive heart failure (CHF). Aldosteronism is intrinsically coupled to Zn and Ca dyshomeostasis, in which consequent hypozincemia compromises Zn homeostasis and Zn-based antioxidant defenses that contribute to the CHF prooxidant phenotype. Ionized hypocalcemia leads to secondary hyperparathyroidism with parathyroid hormone-mediated Ca overloading of diverse cells, including cardiomyocytes. When mitochondrial Ca overload exceeds a threshold, myocyte necrosis follows. The reciprocal regulation involving cytosolic free [Zn]i as antioxidant and [Ca]i as prooxidant can be uncoupled in favor of Zn-based antioxidant defenses. Increased [Zn]i acts as a multifaceted antioxidant by: (1) inhibiting Ca entry through L-type channels and hence cardioprotectant from the Ca-driven mitochondriocentric signal-transducer effector pathway to nonischemic necrosis, (2) serving as catalytic regulator of Cu/Zn-superoxide dismutase, and (3) activating its cytosolic sensor, metal-responsive transcription factor that regulates the expression of relevant antioxidant defense genes. Albeit present in subnanomolar range, increased cytosolic free [Zn]i enhances antioxidant capacity that confers cardioprotection. It can be achieved exogenously by ZnSO4 supplementation or endogenously using a ß3-receptor agonist (eg, nebivolol) that enhances NO generation to release inactive cytosolic Zn bound to metallothionein. By recognizing the pathophysiologic relevance of Zn dyshomeostasis in the prooxidant CHF phenotype and by exploiting the pharmacophysiologic potential of [Zn]i as antioxidant, vulnerable cardiomyocytes under assault from neurohormonal activation can be protected and the myocardium spared from adverse structural remodeling.

Prevention of liver fibrosis by triple helix-forming oligodeoxyribonucleotides targeted to the promoter region of type I collagen gene.

Hepatic fibrosis leading to cirrhosis remains a global health problem. The most common etiologies are alcoholism and viral infections. Liver fibrosis is associated with major changes in both quantity and composition of extracellular matix and leads to disorganization of the liver architecture and irreversible damage to the liver function. As of now there is no effective therapy to control fibrosis. The end product of fibrosis is abnormal synthesis and accumulation of type I collagen in the extracellular matrix, which is produced by activated stellate or Ito cells in the damaged liver. Therefore, inhibition of transcription of type I collagen should in principle inhibit its production and accumulation in liver. Normally, DNA exists in a duplex form. However, under some circumstances, DNA can assume triple helical (triplex) structures. Intermolecular triplexes, formed by the addition of a sequence-specific third strand to the major groove of the duplex DNA, have the potential to serve as selective gene regulators. Earlier, we demonstrated efficient triplex formation between the exogenously added triplex-forming oligodeoxyribonucleotides (TFOs) and a specific sequence in the promoter region of the COL1A1 gene. In this study we used a rat model of liver fibrosis, induced by dimethylnitrosamine, to test whether these TFOs prevent liver fibrosis. Our results indicate that both the 25-mer and 18-mer TFOs, specific for the upstream nucleotide sequence from -141 to -165 (relative to the transcription start site) in the 5' end of collagen gene promoter, effectively prevented accumulation of liver collagen and fibrosis. We also observed improvement in liver function tests. However, mutations in the TFO that eliminated formation of triplexes are ineffective in preventing fibrosis. We believe that these TFOs can be used as potential antifibrotic therapeutic molecules.

PKC eta regulates occludin phosphorylation and epithelial tight junction integrity.

PKC eta is expressed predominantly in the epithelial tissues; however, its role in the regulation of epithelial tight junctions (TJs) is unknown. We present evidence that PKC eta phosphorylates occludin on threonine residues (T403 and T404) and plays a crucial role in the assembly and/or maintenance of TJs in Caco-2 and MDCK cell monolayers. Inhibition of PKC eta by specific pseudo substrate inhibitor or knockdown of PKC eta by specific shRNA disrupts the junctional distribution of occludin and ZO-1 and compromises the epithelial barrier function. Expression of dominant negative, PKC eta(K394R) disrupts the TJ and barrier function, whereas wild-type PKC eta and constitutively active PKC eta(A161E) enhance the TJ integrity. Inhibition and knockdown of PKC eta or expression of PKC eta(K394R) induce dephosphorylation of occludin on threonine residues, whereas active PKC eta elevates occludin phosphorylation. PKC eta directly interacts with the C-terminal domain of occludin and phosphorylates it on highly conserved T403 and T404. T403/404A mutations result in the loss of occludin's ability to localize at the TJs, whereas T403/404D mutations attenuates the PKC eta inhibitor-mediated redistribution of occludin from the intercellular junctions. These results reveal an important mechanism of epithelial TJ regulation by PKC eta.

The N-terminal domain of y-box binding protein-1 induces cell cycle arrest in g2/m phase by binding to cyclin d1.

Y-box binding protein YB-1 is a multifunctional protein involved in cell proliferation, regulation of transcription and translation. Our previous study indicated that disruption of one allele of Chk-YB-1b gene in DT-40 cells resulted in major defects in the cell cycle. The abnormalities seen in heterozygous mutants could be attributed to a dominant negative effect exerted by the disrupted YB-1 allele product. To test this hypothesis the N-terminal sequence of the YB-1 was fused with the third helix of antennapedia and the green fluorescent protein. These purified fusion proteins were introduced into rat hepatoma cells and their effect on cell proliferation was studied. Results indicate that the N-terminal 77 amino acid domain of the YB-1 protein induced the cells to arrest in G2/M phase of the cell cycle and undergo apoptosis. Additional deletion analysis indicated that as few as 26 amino acids of the N-terminus of YB-1 can cause these phenotypic changes. We further demonstrated that this N-terminal 77 amino acid domain of YB-1 sequesters cyclin D1 in the cytoplasm of cells at G2/M phase of cell cycle. We conclude that the N-terminal domain of YB-1 plays a major role in cell cycle progression through G2/M phase of cell cycle.

Sequence-specific triple helix formation with genomic DNA.

We have previously demonstrated site-specific delivery of antiparallel phosphorothioate triplex forming oligonucleotide (TFO) specific to -165 to -141 promoter region of alpha1(I) collagen (abbreviated as APS165) to hepatic stellate cells (HSCs) of fibrotic rats after conjugation with mannose 6-phosphate-bovine serum albumin. However, we still need to determine whether there is correlation between transcription inhibition and triplex formation with genomic DNA. In this study, APS165 was modified with psoralen and the extent of triplex formation with alpha1(I) collagen DNA was determined in naked genomic DNA, isolated nuclei of HSC-T6 cells and whole cells by using a simple real-time PCR based method. In this method, a purification step was added to remove unbound APS165, which eliminated the possible artifacts during real-time PCR. Psoralen photoadduct formation was shown to be essential to retain triplex structure under denaturing conditions. On naked genomic DNA, 82.2% of DNA formed triplex and 36.7% of genomic DNA in isolated nuclei at 90 min contained triplex structure. As quantified by real-time PCR, 50% of genomic DNA in living cells at 12 h postincubation contained triplex structures. Furthermore, the triplex formation was dose-dependent with 26.5% and 50% of DNA having triplex structure at concentrations of 1 microM and 5 microM, respectively. Moreover, on a plasmid pCol-CAT220 containing rat alpha1(I) gene promoter (-225 to +113), 75.3% of triplex formation was observed, which was correlated with a 73.6% of transcription inhibition. These findings will further strengthen the therapeutic applications of APS165.

Receptor-mediated hepatic uptake of M6P-BSA-conjugated triplex-forming oligonucleotides in rats.

Excessive production of extracellular matrix, predominantly type I collagen, results in liver fibrosis. Earlier we synthesized mannose 6-phosphate-bovine serum albumin (M6P-BSA) and conjugated to the type I collagen specific triplex-forming oligonucleotide (TFO) for its enhanced delivery to hepatic stellate cells (HSCs), which is the principal liver fibrogenic cell. In this report, we demonstrate a time-dependent cellular uptake of M6P-BSA-33P-TFO by HSC-T6 cells. Both cellular uptake and nuclear deposition of M6P-BSA-33P-TFO were significantly higher than those of 33P-TFO, leading to enhanced inhibition of type I collagen transcription. Following systemic administration into rats, hepatic accumulation of M6P-BSA-33P-TFO increased from 55% to 68% with the number of M6P per BSA from 14 to 27. Unlike 33P-TFO, there was no significant decrease in the hepatic uptake of (M6P)20-BSA-33P-TFO in fibrotic rats. Prior administration of excess M6P-BSA decreased the hepatic uptake of (M6P)20-BSA-33P-TFO from 66% to 40% in normal rats, and from 60% to 15% in fibrotic rats, suggesting M6P/insulin-like growth factor II (M6P/IGF II) receptor-mediated endocytosis of M6P-BSA-33P-TFO by HSCs. Almost 82% of the total liver uptake in fibrotic rats was contributed by HSCs. In conclusion, by conjugation with M6P-BSA, the TFO could be potentially used for the treatment of liver fibrosis.

Enhanced hepatic uptake and bioactivity of type alpha1(I) collagen gene promoter-specific triplex-forming oligonucleotides after conjugation with cholesterol.

A triplex-forming oligonucleotide (TFO) specific for type alpha1(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 (33)P-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 alpha1(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 (33)P-TFO-Chol after systemic administration was 72.22% of the dose compared with 45.8% of (33)P-TFO. There was significant increase in the uptake of (33)P-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.

High prevalence of hepatitis C virus infection and genotype distribution among general population, blood donors and risk groups.

The prevalence of hepatitis C virus (HCV) infection in the general population and in various high risk groups in south India was assessed. A total of 258 out of 3589 (7.1%) subjects (both general and risk groups) tested positive for HCV RNA by RT-PCR, while the third generation ELISA detected only 6.1% (221/3589). This suggests that a number of cases go unreported, as screening of blood and blood products is done primarily by ELISA. Among 124 chronic renal failure (CRF) patients with a history of renal transplant or haemodialysis, 37% were found to be positive for HCV RNA by RT-PCR. We also found a significantly higher rate of transmission of HCV among people exposed to tattooing (2.8%) and pilgrims (5.8%) (slashing a cultural practice in one sect of Muslims). In addition, our studies also reveal a high prevalence of HCV infection (44%) among patients with Lichen planus. The most prevalent genotype observed in our population was 1b (43.4%) followed by 3b (30.2%). The other genotype 1a was observed in 16.6% of patients followed by 3a observed in 3.4% of the patients. Our findings suggest that HCV may be the major cause of post-transplant hepatitis in Indian patients with CRF and indicate the necessity for stringent screening procedures for these viral infections.

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.

Expression of the multifunctional Y-box protein, YB-1, in myofibroblasts of the infarcted rat heart.

Intracellular signaling mechanisms regulating the turnover of alpha-SMA-positive myofibroblasts (myoFbs) at the site of myocardial infarction (MI) are poorly understood. Y-Box (YB)-1, a multifunctional protein, may be involved in regulation of proliferation, migration and apoptosis of myoFbs. Our objective was to study the expression of YB-1 in the infarcted rat heart and its localization in myoFbs. On days 3-28 following MI, we monitored YB-1 expression and its colocalization with alpha-SMA, and proliferation markers PCNA and Ki-67 in infarcted tissue by Western blot, immunohistochemistry, and immunofluorescent double-labeling. YB-1 is barely detectable in normal myocardium. At the infarct site, however, YB-1 is markedly elevated from day 3 post-MI concomitant with the induction of cell proliferation. MyoFbs are the major source of YB-1 and retain it up to day 28 post-MI. We suggest early expression of YB-1 promotes proliferation and migration of myoFbs, whereas prolonged expression may be responsible for scar formation.

Biodistribution and hepatic uptake of triplex-forming oligonucleotides against type alpha1(I) collagen gene promoter in normal and fibrotic rats.

Fibrosis is characterized by excessive production of extracellular matrix (ECM) components, predominantly type 1 collagen. Earlier we developed an antigene approach, using a type alpha1(I) promoter specific TFO to inhibit collagen gene expression. In this report, biodistribution and hepatic cellular and subcellular localization of the 25-mer antiparallel phosphorothioate triplex-forming oligonucleotide (APS TFO) were determined after intravenous injection into rats. TFOs distributed to all the major organs, with higher uptake in the liver, kidney, and spleen. The plasma concentration versus time profile of the (33)P-TFO was biphasic, with 4.36 min as t(1/2)(alpha) of distribution and 34.6 min as t(1/2)(beta) of elimination. TFO concentrations in the liver increased nonlinearly with increase in its dose from 0.2 to 50 mg/kg, but decreased when injected into fibrotic rats. Competition studies with polyinosinic acid (polyI) and dextran sulfate suggested the involvement of scavenger receptors in the hepatic uptake of the TFO. Intrahepatic cellular distribution by Kupffer, endothelial, and hepatic stellate cells (HSCs) accounted for almost 70% of the liver uptake of (33)P-TFO, while only 30% was associated with hepatocytes. The level of liver nuclei-associated TFO was much lower relative to that found in the cytoplasm at 2 and 4 h postinjection. TFO, however, inhibited collagen expression as evidenced by Sirius red staining of the liver section of fibrotic rats. In conclusion, systemic delivery of the TFO against type alpha1(I) collagen gene promoter may be used for the treatment of liver fibrosis.

Targeted delivery of a triplex-forming oligonucleotide to hepatic stellate cells.

Liver fibrosis is characterized by abnormal accumulation of extracellular matrix (ECM), namely, fibrillar collagens in the hepatic stellate cells (HSCs). Earlier, we developed an antigene approach, using a type alpha1(I) collagen gene promoter specific triplex-forming oligonucleotide (TFO) to inhibit collagen gene expression. In this paper, to enhance overall delivery of TFOs to the liver and more specifically to HSCs, we synthesized mannose 6-phosphate-bovine serum albumin (M6P-BSA) by phosphorylating p-nitrophenyl-alpha-d-mannopyranoside, reducing its nitro group, and reacting it with thiophosgene to produce p-isothiocyanatophenyl-6-phospho-alpha-d-mannopyranoside (itcM6P) for conjugation with BSA. (33)P-TFO was conjugated with M6P-BSA via a disulfide bond, and the stability of the (M6P)(20)-BSA-TFO conjugate was determined. Following tail vein injection into rats, (M6P)(20)-BSA-(33)P-TFO rapidly cleared from the circulation and accumulated mainly in the liver. Almost 66% of the injected (M6P)(20)-BSA-(33)P-TFO accumulated in the liver at 30 min postinjection, which was significantly higher than that deposited after injection of (33)P-TFO. A large proportion of the injected (M6P)(20)-BSA-(33)P-TFO was taken up by the HSCs as evidenced by determination of radioactivity in the digested liver cells upon liver perfusion and separation on a Nycodenz gradient. Therefore, this TFO conjugate may be used for the treatment of liver fibrosis.

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.

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.

Targeted disruption of one allele of the Y-box protein gene, Chk-YB-1b, in DT40 cells results in major defects in cell cycle.

Y-box or inverted CCAAT box-binding proteins are multifunctional regulators of transcription and translation of several genes. Although YB-1 has been shown to play a key role in cell cycle, to date, there is no direct evidence. We disrupted one allele of Chk-YB-1b in a chicken pre-B lymphocyte cell line, DT40. Compared to wild-type DT40 cells, these heterozygous DT40YB1b(+/-) cells with one copy of the wild-type Chk-YB-1b allele showed multiple abnormalities, which include slower rate of growth, abnormal cell morphology, increased cell size, and increased genomic DNA content. These phenotypic defects resemble those cells that have a block in G2 and/or mitosis (G2/M). In addition, we have observed that a fraction of these heterozygous DT40YB1b(+/-) cells undergo apoptosis. In conclusion, we have discovered major defects in the G2/M phase of cell cycle in YB-1 knocked-out heterozygous mutant cells, providing for the first time direct evidence establishing a crucial role for YB-1 in cell proliferation.