Current therapies for diseases of heart muscle (cardiomyopathy) and aorta (aortopathy) include inhibitors of the renin-angiotensin system, β-adrenergic antagonists, and the statin class of cholesterol-lowering agents. These therapies have limited efficacy, as adverse cardiovascular events continue to occur with some frequency in patients taking these drugs. Although cardiomyopathy and aortopathy can coexist in a number of conditions (for example, Marfan's syndrome, acromegaly, pregnancy, and aging), pathogenetic molecular links between the two Copyright 2010 by the American Association for the Advancement of Science; all rights reserved. † To whom correspondence should be addressed. mukesh.jain2@case.edu. * These authors contributed equally to this work. SUPPLEMENTARY MATERIALwww.sciencetranslationalmedicine.org/cgi/content/full/2/26/26ra26/DC1 Fig. S1. Cardiovascular abnormalities in AngII-treated mice and cultured cells. Fig. S2. Baseline abnormalities in Klf15 −/− heart and aorta. Fig. S3. Cardiac mass and systolic blood pressure after AngII infusion. Fig. S4. Histologic parameters in aortas. Fig. S5. MMP-3 abundance in aortic smooth muscle. Fig. S6. p53 mRNA concentrations in heart and aortic tissue. Fig. S7. p300 abundance in hearts, curcumin administration protocol, and aortic morphometry after curcumin therapy. Table S1. Baseline cardiac parameters in Klf15 −/− and wild-type mice. Table S2. Cardiac parameters in Klf15 −/− and wild-type mice after AngII infusion. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript diseases remain poorly understood. We reasoned that identification of common molecular perturbations in these two tissues could point to therapies for both conditions. Here, we show that deficiency of the transcriptional regulator Kruppel-like factor 15 (Klf15) in mice leads to both heart failure and aortic aneurysm formation through a shared molecular mechanism. Klf15 concentrations are markedly reduced in failing human hearts and in human aortic aneurysm tissues. Mice deficient in Klf15 develop heart failure and aortic aneurysms in a p53-dependent and p300 acetyltransferase-dependent fashion. KLF15 activation inhibits p300-mediated acetylation of p53. Conversely, Klf15 deficiency leads to hyperacetylation of p53 in the heart and aorta, a finding that is recapitulated in human tissues. Finally, Klf15-deficient mice are rescued by p53 deletion or p300 inhibition. These findings highlight a molecular perturbation common to the pathobiology of heart failure and aortic aneurysm formation and suggest that manipulation of KLF15 function may be a productive approach to treat these morbid diseases.

SUMMARY Diurnal variation in nitrogen homeostasis is observed across phylogeny. But whether these are endogenous rhythms, and if so, molecular mechanisms that link nitrogen homeostasis to the circadian clock remain unknown. Here, we provide evidence that a clock-dependent peripheral oscillator, Krüppel-like factor15 transcriptionally coordinates rhythmic expression of multiple enzymes involved in mammalian nitrogen homeostasis. In particular, Krüppel-like factor15-deficient mice exhibit no discernable amino acid rhythm, and the rhythmicity of ammonia to urea detoxification is impaired. Of the external cues, feeding plays a dominant role in modulating Krüppel-like factor15 rhythm and nitrogen homeostasis. Further, when all behavioral, environmental and dietary cues were controlled in humans, nitrogen homeostasis still expressed endogenous circadian rhythmicity. Thus, in mammals, nitrogen homeostasis exhibits circadian rhythmicity, and is orchestrated by Krüppel-like factor15.

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Activation of vascular endothelial growth factor (VEGF) receptor-3 (VEGFR-3) by VEGF-C initiates lymphangiogenesis by promoting lymphatic proliferation and migration. However, it is unclear whether VEGFR-3 signaling is required beyond these initial stages, namely during the organization of new lymphatic endothelial cells (LECs) into functional capillaries. Furthermore, the role of VEGFR-2, which is also expressed on LECs and binds VEGF-C, is unclear. We addressed these questions by selectively neutralizing VEGFR-3 and/or VEGFR-2 for various time periods in an adult model of lymphangiogenesis in regenerating skin. While blocking either VEGFR-2 or VEGFR-3 with specific antagonist mAbs (DC101 and mF4-31C1, respectively) prior to lymphatic migration prevented lymphangiogenesis, blocking VEGFR-3 subsequent to migration did not affect organization into functional capillaries, and VEGFR-2 blocking had only a small hindrance on organization. These findings were confirmed in vitro using human LECs and anti-human antagonist mAbs (IMC-1121a and hF4-3C5): both VEGFR-2 and -3 signaling were required for migration and proliferation, but tubulogenesis in 3D cultures was unaffected by VEGFR-3 blocking and partially hindered by VEGFR-2 blocking. Furthermore, both in vitro and in vivo, while VEGFR-3 blocking had no effect on LEC organization, coneutralization of VEGFR-2, and VEGFR-3 completely prevented lymphatic organization. Our findings demonstrate that cooperative signaling of VEGFR-2 and -3 is necessary for lymphatic migration and proliferation, but VEGFR-3 is redundant with VEGFR-2 for LEC organization into functional capillaries.

Krüppel-like factors (KLF) are a subfamily of the zinc-finger class of transcriptional regulators that play important roles in diverse cellular processes. While a number of KLFs are expressed in cardiomyocytes, little is known about their specific roles in the heart in vivo. Here, we demonstrate that KLF4 is induced by hypertrophic stimuli in cultured cardiomyocytes and in the mouse heart. Overexpression of KLF4 in neonatal rat ventricular myocytes inhibits three cardinal features of cardiomyocyte hypertrophy: fetal gene expression, protein synthesis, and cell enlargement. Conversely, mice with cardiomyocyte-specific deletion of KLF4 (CM-K4KO) are highly sensitized to transverse aortic constriction (TAC) and exhibit high rates of mortality. CM-K4KO mice that survive TAC display severe pathologic cardiac hypertrophy characterized by increased cardiac mass, depressed LV systolic function, pulmonary congestion, cavity dilation and attenuated LV wall thickening when compared to control genotypes. In addition, CM-K4KO mice develop increased myocardial fibrosis and apoptotic cell death after TAC. Collectively, these studies implicate KLF4 as a novel transcriptional regulator that is indispensible for the hearts response to stress in vivo.

Objective A central function of the endothelium is to serve as a selective barrier that regulates fluid and solute exchange. While perturbation of barrier function can contribute to numerous disease states, our understanding of the molecular mechanisms regulating this aspect of endothelial biology remains incompletely understood. Accumulating evidence implicates the Kruppel-like factor 2 (KLF2) as a key regulator of endothelial function. However, its role in vascular barrier function is unknown. Methods and Results To assess the role of KLF2 in vascular barrier function in vivo, we measured the leakage of Evans Blue dye into interstitial tissues of the mouse ear after treatment with mustard oil. By comparison to KLF2+/+ mice, KLF2+/− mice exhibited a significantly higher degree of vascular leak. In accordance with our in vivo observation, adenoviral overexpression of KLF2 in HUVECs strongly attenuated the increase of endothelial leakage by thrombin and H2O2 as measured by FITC-Dextran passage. Conversely, KLF2 deficiency in HUVECs and primary endothelial cells derived from KLF2+/− mice exhibited a marked increase in thrombin and H2O2-induced permeability. Mechanistically, our studies indicate that KLF2 confers barrier-protection via differential effects on the expression of key junction protein occludin and modification of a signaling molecule (myosin light chain) that regulate endothelial barrier integrity. Conclusions These observations identify KLF2 as a novel transcriptional regulator of vascular barrier function.

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