The recent study by Cunningham, Lewis, Curnow, Glazner, and Massie [1] on the attitudes of respiratory physicians and clinic coordinators towards cystic fibrosis (CF) carrier screening drew several unsupported conclusions because the α level of 0.05 was not corrected for the large number of hypothesis tests conducted, leading to a Type 2 error and the acceptance of hypotheses that were likely false [2].

A recent study by Leonard, Lebecque, Dingemanse, and Leal [1] tested the effect of Miglustat, an alpha inhibitor on the cystic fibrosis conductance regulator gene using total chloride secretion in the nasal epithelium as the key variable estimated from basal nasal potential differences. The conclusion was drawn that “There was no evidence of a treatment effect on any nasal potential difference variable.” This conclusion may not be correct because of a slight misinterpretation of their statistical results. There also is a question of whether longer exposure periods than 8 days would have produced a more pronounced effect.

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Thesis: M.S., Massachusetts Institute of Technology, Department of Nutrition and Food Science, 1980 / Includes bibliographical references. / by Thomas Henry Openshaw. / M.S. / M.S. Massachusetts Institute of Technology, Department of Nutrition and Food Science

Transplanted embryonic stem (ES) cells following myocardial infarction (MI) contribute to limited cardiac repair and regeneration with improved function. Therefore novel strategies are still needed to enhance the efficacy by which ES cells differentiate into cardiac cell types and inhibit adverse remodeling in the infarcted myocardium. Our studies evaluate whether genetic manipulation of transplanted ES cells employing miR- 1, a pro-cardiac microRNA, and TIMP-1, an anti-apoptotic and anti-fibrotic protein, will enhance cardiac myocyte differentiation, inhibit native cardiac apoptosis, and reduce fibrosis in the infarcted myocardium. Furthermore, we assess levels of associated pro- (caspase-3, PTEN) and anti-(Akt) apoptotic proteins as well as a pro-fibrotic protein (MMP-9) in the post-MI and cell transplanted heart. microRNAs (miRs) have emerged as critical regulators of various physiological processes including development, differentiation, metabolism, and death. Indeed, miR- 1 plays an integral role in early cardiac development in Drosophila and mice as well as mediates differentiation of cardiac myocytes in vitro. To that end, we generated ES cells overexpressing miR-1 (miR-1-ES cells), transplanted them into the infarcted myocardium, and evaluated their impact on cardiac myocyte differentiation, myocardial repair, and left ventricular dysfunction post-MI. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared to ES cell and culture media transplanted hearts. Assessment of apoptosis revealed overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-Akt and inhibition of caspase-3, PTEN, and superoxide anion production. A significant reduction iv in interstitial and vascular fibrosis was quantified in miR-1-ES and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1- ES cell and ES cell groups was observed. Finally mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls. Our data suggests miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI ultimately giving rise to enhanced cardiac repair, regeneration, and function. Next, we assessed the role of miR-1-ES cells in a chronic model of MI as research has shown that apoptosis occurs not only hours but months following ischemia. 4 weeks following transplantation into the infarcted myocardium, we provide evidence demonstrating reduced cardiac apoptosis in miR-1-ES cell transplanted hearts compared to respective controls. Moreover, we show significant elevation of p-Akt levels and diminished PTEN levels in hearts transplanted with miR-1-ES cells as determined by enzyme-linked immunoassays. Finally, using echocardiography, we reveal mice receiving miR-1-ES cell transplantation post-MI had significantly improved cardiac function compared with animals transplanted with ES cell and culture media. Our data suggests that miR-1, when overexpressed in transplanted ES cells, has the capacity to inhibit apoptosis long term while attenuating contractility loss. In addition to enhancing cardiac-specific donor cell differentiation, improving the efficacy by which stem cells promote cell survival and repair in the host myocardium is imperative in the pursuit of refining and optimizing stem cell therapy. To that end, we overexpressed TIMP-1, an endogenous inhibitor of apoptosis and fibrosis, in ES cells (TIMP-1-ES cells), transplanted them into infarcted myocardium, and evaluated their v impact on adverse cardiac remodeling. Immunofluorescence, TUNEL staining, caspase-3 activity, ELISAs, histology, and echocardiography were used to assess apoptosis, fibrosis, and heart function. Hearts transplanted with TIMP-1-ES cells demonstrated a reduction in apoptosis as well as an increase in p-Akt activity compared with ES cells or culture media controls. Interstitial and vascular fibrosis was significantly decreased in the TIMP-1-ES cell group compared to controls. Furthermore, MMP-9, a key pro-fibrotic protein, was significantly reduced following TIMP-1-ES cell transplantation. Echocardiography data showed fractional shortening and ejection fraction were significantly improved in the TIMP-1-ES cell group compared with respective controls. Our data suggest that transplanted ES cells overexpressing TIMP- 1 attenuate adverse myocardial remodeling and improve cardiac function compared with ES cells. Overall, our data suggest that genetic manipulation of ES cells following transplantation in the infarcted heart enhances cardiac myocyte differentiation, inhibits apoptosis and fibrosis as well as improves cardiac function.

Background: Vascular stiffening develops with both hypertension and aging, and is a strong predictor of end-organ damage. Excessive deposition of collagen by vascular smooth muscle cells (VSMCs) can lead to decreased compliance of vessels such as the aorta. The IRE1α arm of the unfolded protein response is activated in cells with a secretory phenotype due to its role in augmenting protein folding capacity. We hypothesize that by a similar mechanism, VSMCs transitioning to a collagen-secreting phenotype in response to TGF-β1 require the activation of IRE1. Inhibition of this pathway is hypothesized to reduce collagen secretion and hence prevent the development of fibrosis in the aorta. Methods: Collagen deposition by VSMCs in vitro was measured using immunoblotting and a Picrosirius Red-based colorimetric assay. Western blot and qRT-PCR were used to assess the expression of ER stress markers. Ex vivo culture of aortic rings was also performed to determine the effect of 4µ8c on TGF-β1-induced vascular stiffening. 12-14 week old male spontaneously hypertensive rats were divided into three treatment groups: 1) No treatment, 2) L-NAME (50 mg/L), and 3) L-NAME and the IRE1α inhibitor 4µ8c (2.5 mg/kg/day i.p.). Aortic compliance after 18 days of treatment was measured ex vivo using a wire myograph to construct tension-diameter curves. Results: Inhibition of IRE1α endonuclease activity by 4µ8c reduced collagen production in VSMCs stimulated with TGF-β1 or Ang II. A decrease in the expression of the collagen-associated chaperones PDI, GRP78 and GRP94 was observed. Aortic rings treated with TGF-β1 developed vascular stiffening, which was improved by co-treatment with 4µ8c. SHRs treated with L-NAME for 18 days developed aortic stiffening, which was prevented by daily injections of 4µ8c. Conclusions: Our data suggest that inhibition of the IRE1α pathway can reduce vascular stiffening and fibrosis by disrupting the collagen biosynthesis pathway in VSMCs. / Thesis / Master of Science (MSc)

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Indiana University-Purdue University Indianapolis (IUPUI) / Hepatic fibrosis is a cellular repair mechanism that is initiated upon prolonged damage to the liver, resulting in an accumulation of excess extracellular matrix. This eventually leads to the formation of scar tissue, which disrupts the hepatic architecture and causes liver dysfunction. Hepatic stellate cells (HSCs) play a major role in hepatic fibrosis. However, the molecular mechanisms remain incompletely understood. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ or WWTR1), key players of the Hippo pathway, have been implicated in the liver fibrosis, but the HSC-specific functions of YAP and TAZ are largely unclear. Here we have identified Sirtuin 6 (SIRT6), an NAD+ dependent deacetylase, as a key epigenetic regulator in the protection against hepatic fibrosis by suppressing the YAP/TAZ activity. SIRT6 has been previously implicated in the regulation of the canonical transforming growth factor β (TGFβ)-SMAD3 pathway. This study has revealed the significant contribution of the non-canonical pathways including the Hippo pathway to the development of hepatic fibrosis. HSC-specific Sirt6 deficient mice developed severe fibrosis when fed a high-fat-cholesterol-cholate diet compared to their wild-type counterparts. YAP became more active in the SIRT6-deficient HSCs. Expression of the YAP/TAZ downstream genes like CTGF, CYR61 and ANKRD1 were elevated in the SIRT6-deficient HSCs. Biochemical and mutagenic analyses have revealed that SIRT6 deacetylates YAP and TAZ at key lysine residues and reprograms the composition of the TEA domain transcription factor complex to suppress the YAP/TAZ function in the hepatic fibrogenesis.