Wnt Signaling Interactor WTIP (Wilms Tumor Interacting Protein) Underlies Novel Mechanism for Cardiac Hypertrophy.

BACKGROUND: The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. METHODS: In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell-derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. RESULTS: WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio. Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell-derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. CONCLUSIONS: We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.

Epitope Spreading and the Efficacy of Immune Checkpoint Inhibition in Cancer.

Therapeutic antibodies that target immune checkpoints have revolutionized cancer therapy. While these checkpoints restrain T cell activation in response to antigen engagement, checkpoint inhibitors de-repress such tumor-associated T cells, and have generated major clinical responses in multiple tumor types. Nonetheless, the vast majority of cancers remain resistant to this therapeutic approach as currently deployed, either through intrinsic or acquired resistance mechanisms. One key question involves the identity of the tumor targets which effector T cells recognize. Tumor-specific mutant epitopes (often called neoantigens) represent a favored example, whose recognition has been demonstrated in certain contexts. While potentially helpful in identifying likely therapeutic opportunities (such as cancers harboring DNA repair defects), numerous cancers are relatively deficient in neoantigen loads. This commentary discusses the prospect that a phenomenon of "epitope spreading" may occur in certain high mutation contexts, giving rise to T cell responses against non-mutated/wild-type lineage proteins. Recent evidence is also discussed that suggests this mechanism may be exploited to purposely trigger epitope spreading and induce systemic tumor eradication in neoantigen-deficient cancers.

Silencing of MYH7 ameliorates disease phenotypes in human iPSC-cardiomyocytes.

Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy (HCM) to date have focused on mouse models of disease. We here examine allele-specific silencing in a human-cell model of HCM. We investigate two methods of silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategy's effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. These findings are the first exploration of allele-specific silencing in a human HCM model and provide a foundation for further exploration of silencing as a therapeutic treatment for MYH7-mutation-associated cardiomyopathy.

Signaling pathways and immune evasion mechanisms in classical Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) is an unusual B-cell-derived malignancy in which rare malignant Hodgkin and Reed-Sternberg (HRS) cells are surrounded by an extensive but ineffective inflammatory/immune cell infiltrate. This striking feature suggests that malignant HRS cells escape immunosurveillance and interact with immune cells in the cancer microenvironment for survival and growth. We previously found that cHLs have a genetic basis for immune evasion: near-uniform copy number alterations of chromosome 9p24.1 and the associated PD-1 ligand loci, CD274/PD-L1 and PDCD1LG2/PD-L2, and copy number-dependent increased expression of these ligands. HRS cells expressing PD-1 ligands are thought to engage PD-1 receptor-positive immune effectors in the tumor microenvironment and induce PD-1 signaling and associated immune evasion. The genetic bases of enhanced PD-1 signaling in cHL make these tumors uniquely sensitive to PD-1 blockade.

Signaling pathways and immune evasion mechanisms in classical Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) is an unusual B-cell-derived malignancy in which rare malignant Hodgkin and Reed-Sternberg (HRS) cells are surrounded by an extensive but ineffective inflammatory/immune cell infiltrate. This striking feature suggests that malignant HRS cells escape immunosurveillance and interact with immune cells in the cancer microenvironment for survival and growth. We previously found that cHLs have a genetic basis for immune evasion: near-uniform copy number alterations of chromosome 9p24.1 and the associated PD-1 ligand loci, CD274/PD-L1 and PDCD1LG2/PD-L2, and copy number-dependent increased expression of these ligands. HRS cells expressing PD-1 ligands are thought to engage PD-1 receptor-positive immune effectors in the tumor microenvironment and induce PD-1 signaling and associated immune evasion. The genetic bases of enhanced PD-1 signaling in cHL make these tumors uniquely sensitive to PD-1 blockade.

Synergistic activation of extracellular signal-regulated kinase in human dermal fibroblasts by human telomerase reverse transcriptase and transforming growth factor-beta1.

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is primarily known for its ability to elongate telomeres for maintaining chromosomal integrity and delaying cellular senescence. Recently, hTERT has emerged as having a role in promoting cellular proliferation that is independent of telomere elongation. How hTERT elicits this novel function is a fundamental question in cell biology. Understanding this question may have therapeutic implications in regenerative medicine for patients with damaged organs or tissues, cardiovascular disorders, stroke, ischemic chronic wounds, and other ischemia-reperfusion injuries. Toward this end, we treated hTERT-transfected human dermal fibroblasts (HDFs) with transforming growth factor (TGF)-beta1 and investigated the activation of extracellular signal-regulated kinase (ERK) 1/2, vital mediators of cell proliferation. MATERIALS AND METHODS: Primary HDFs were transfected with either recombinant adenovirus expressing hTERT (Ad-hTERT) or control adenovirus (Ad-NULL) and subsequently treated with TGF-beta1 (2 pg/mL). ERK 1/2 activation was determined by Western blotting using an antibody recognizing only activated ERK 1/2 that is dually phosphorylated at Thr(202) and Tyr(204). TGF-beta1, TGFbeta-RI, TGFbeta-RII, and Col1 A1 mRNA levels were analyzed by real-time PCR. RESULTS: Ad-hTERT-transfected HDFs showed more than 7-fold up-regulation of phospho-ERK 1/2 over Ad-NULL-transfected HDFs upon TGF-beta1 treatment. The synergistic ERK 1/2 activation in Ad-hTERT-transfected HDFs occurred as early as 10 min and was sustained for at least 30 min after TGF-beta1 treatment. There were no statistically significant differences in TGF-beta1, TGFbeta-RI, TGFbeta-RII, and Col1 A1 mRNA levels between HDFs that were transfected with Ad-hTERT and those that were transfected with Ad-NULL after TGF-beta1 treatment. CONCLUSIONS: hTERT and extremely low concentrations of TGF-beta1 (2 pg/mL) synergistically activate ERK 1/2 in HDFs by a mechanism that is independent of the autocrine TGF-beta1 loop.

Reduced up-regulation of cytoprotective genes in rat cutaneous tissue during the second cycle of ischemia-reperfusion.

Chronic wounds are major health problems that affect millions of people in the United States every year. Management of these wounds costs billions of dollars annually in the United States. Despite their clinical importance, the molecular mechanisms underlying these clinical conditions remain elusive. Repetitive ischemia-reperfusion (I-R) may play a pivotal role in chronic wound formation. The development of therapies for these wounds is hindered by the lack of animal models that allow identification of the molecular mechanisms underlying chronic wound formation. In the first study of its kind, we adapted our rat pressure sore model by imposing two cycles of ischemia (2 hours) and two cycles of reperfusion (24 hours), and we examined gene expression to better understand the molecular events that occur at the very early stages of cutaneous I-R injury with a goal of devising preventing strategies. We successfully tested our hypothesis and demonstrated that while cytoprotective genes, such as heat shock protein 70, heat shock protein 90, hypoxia-inducible factor-1alpha, vascular endothelial growth factor, and heme oxygenase-1, were initially up-regulated during the first cycle of I-R, their up-regulation was subsequently reduced or completely abolished during the second cycle of I-R. These findings raise the possibility that reduced up-regulation of these cytoprotective genes may be causally linked to cutaneous I-R injury.

Adenoviral human telomerase reverse transcriptase dramatically improves ischemic wound healing without detrimental immune response in an aged rabbit model.

Chronic ischemic wounds are major clinical problems, and are especially prevalent in elderly patients. Management of these wounds costs billions of dollars annually in the United States. Because of the severe impairment in tissue repair, ischemic wounds among the aged are major challenges for physicians. For example, transforming growth factor-beta1 stimulates healing of young patients' ischemic wounds, but it is totally ineffective in treating the ischemic wounds of aged patients. Therefore, our goal is to develop a better therapeutic strategy for elderly patient ischemic wounds. Because human telomerase reverse transcriptase (hTERT) has emerged as having a role in promoting cell proliferation, we hypothesized that hTERT overexpression may improve ischemic wound healing in the elderly. We successfully tested this hypothesis by demonstrating for the first time that gene delivery of hTERT by adenovirus (Ad-hTERT) dramatically improved ischemic wound healing in an aged rabbit model. Importantly, our histological data indicate that no deleterious immune response was induced in the aged rabbits. This finding has broad implications for the field of gene therapy because the foremost obstacle in the use of adenoviral vectors for gene therapy is that they provoke strong innate and adaptive immune responses in the host. Moreover, Ad-hTERT significantly improved survival of primary rabbit dermal fibroblasts that were treated with hypoxia and hydrogen peroxide (oxidative stress). This model is clinically relevant because it simulates the ischemia cycle of an ischemia-reperfusion injury, which can lead to stroke, myocardial infarction, and other tissue injuries. We conclude that Ad-hTERT is an effective and novel approach to treating the ischemic wounds of elderly patients.

Age-dependent response of primary human dermal fibroblasts to oxidative stress: cell survival, pro-survival kinases, and entrance into cellular senescence.

A central question in cell biology is how cells become senescent. After a finite number of cell divisions, normal cultured human cells enter a state of irreversible growth arrest, termed "replicative senescence." Alternatively, oxidative stress in the form of hydrogen peroxide (H(2)O(2)) can render human dermal fibroblasts (HDFs) nonproliferative and quiescent, a phenomenon known as stress-induced premature senescence (SIPS). Although critical to the understanding of the pathophysiological basis of many diseases, there is no research to date that has simultaneously examined the interactions between age, oxidative stress, and SIPS. Therefore, the goals of this study were to examine in concert the interactions between these three factors in primary HDFs, and to test our central hypothesis that aging lowers the ability of primary HDFs to respond to oxidative stress. Our data provide, for the first time, evidence that aging dramatically reduces the capacity of primary HDFs to respond to the challenge of hydrogen peroxide. Specifically, aged HDFs showed decreased cell viability, decreased phosphorylation (activation) of pro-survival kinases (Akt and ERK 1/2), and increased entrance into a senescent state when compared with their younger counterparts. Another important conclusion of this study is that blockade of transforming growth factor-beta1 had a pronounced "rescue effect" in the aged, preventing entrance of HDFs into cellular senescence.

The temporal effects of anti-TGF-beta1, 2, and 3 monoclonal antibody on wound healing and hypertrophic scar formation.

BACKGROUND: A number of studies have implicated transforming growth factor (TGF)-beta1, 2, and 3 (TGF-beta) in wound healing and hypertrophic scarring. We propose that TGF-beta has a temporal effect on these processes. To test this hypothesis, we applied anti-TGF beta1, 2, and 3 monoclonal antibody topically to our dermal ulcer model in the rabbit ear. STUDY DESIGN: Rabbit ear wounds were treated intradermally with anti-TGF-beta1, 2, and 3 antibody at early, middle, and late time points. Treated and untreated control wounds were harvested at various time points and examined histologically to quantify wound healing and scar hypertrophy. Real-time polymerase chain reaction was performed to determine TGF-beta mRNA expression in the treated and control wounds. RESULTS: The early treatment group demonstrated decreased new epithelium and granulation tissue (p < 0.05 versus controls). Scars harvested on days 28 and 40 displayed no difference in scar hypertrophy. Both the middle and late treatment groups demonstrated a significant decrease in scar hypertrophy (p < 0.05). CONCLUSIONS: Treated wounds from the early treatment group displayed delayed wound healing, with no reduction in scar hypertrophy. Later treatment of wounds with the same antibody, beginning 7 days after wounding, resulted in a reduction in scar hypertrophy. These results support our hypothesis and clearly demonstrate that TGF-beta1, 2, and 3 have differential temporal effects during the wound-healing process, and are important for optimal wound healing in the first week after wounding; beyond 1 week, TGF-beta1, 2, and 3 play a critical role in hypertrophic scar formation.