Mice with reduced expression of the telomere‐associated protein Ft1 develop p53‐sensitive progeroid traits

Human AKTIP and mouse Ft1 are orthologous ubiquitin E2 variant proteins involved in telomere maintenance and DNA replication. AKTIP also interacts with A‐ and B‐type lamins. These features suggest that Ft1 may be implicated in aging regulatory pathways. Here, we show that cells derived from hypomorph Ft1 mutant (Ft1^kof/kof) mice exhibit telomeric defects and that Ft1^kof/kof animals develop progeroid traits, including impaired growth, skeletal and skin defects, abnormal heart tissue, and sterility. We also demonstrate a genetic interaction between Ft1 and p53. The analysis of mice carrying mutations in both Ft1 and p53 (Ft1^kof/kof; p53^ko/ko and Ft1^kof/kof; p53^+/ko) showed that reduction in p53 rescues the progeroid traits of Ft1 mutants, suggesting that they are at least in part caused by a p53‐dependent DNA damage response. Conversely, Ft1 reduction alters lymphomagenesis in p53 mutant mice. These results identify Ft1 as a new player in the aging process and open the way to the analysis of its interactions with other progeria genes using the mouse model.     

Notch2 and Notch3 function together to regulate vascular smooth muscle development

Notch signaling has been implicated in the regulation of smooth muscle differentiation, but the precise role of Notch receptors is ill defined. Although Notch3 receptor expression is high in smooth muscle, Notch3 mutant mice are viable and display only mild defects in vascular patterning and smooth muscle differentiation. Notch2 is also expressed in smooth muscle and Notch2 mutant mice show cardiovascular abnormalities indicative of smooth muscle defects. Together, these findings infer that Notch2 and Notch3 act together to govern vascular development and smooth muscle differentiation. To address this hypothesis, we characterized the phenotype of mice with a combined deficiency in Notch2 and Notch3. Our results show that when Notch2 and Notch3 genes are simultaneously disrupted, mice die in utero at mid-gestation due to severe vascular abnormalities. Assembly of the vascular network occurs normally as assessed by Pecam1 expression, however smooth muscle cells surrounding the vessels are grossly deficient leading to vascular collapse. In vitro analysis show that both Notch2 and Notch3 robustly activate smooth muscle differentiation genes, and Notch3, but not Notch2 is a target of Notch signaling. These data highlight the combined actions of the Notch receptors in the regulation of vascular development, and suggest that while these receptors exhibit compensatory roles in smooth muscle, their functions are not entirely overlapping.     

Depletion of Endothelial or Smooth Muscle Cell-Specific Angiotensin II Type 1a Receptors Does Not Influence Aortic Aneurysms or Atherosclerosis in LDL Receptor Deficient Mice

Background

Whole body genetic deletion of AT1a receptors in mice uniformly reduces hypercholesterolemia and angiotensin II-(AngII) induced atherosclerosis and abdominal aortic aneurysms (AAAs). However, the role of AT1a receptor stimulation of principal cell types resident in the arterial wall remains undefined. Therefore, the aim of this study was to determine whether deletion of AT1a receptors in either endothelial cells or smooth muscle cells influences the development of atherosclerosis and AAAs.

Methodology/Principal Findings

AT1a receptor floxed mice were developed in an LDL receptor −/− background. To generate endothelial or smooth muscle cell specific deficiency, AT1a receptor floxed mice were bred with mice expressing Cre under the control of either Tie2 or SM22, respectively. Groups of males and females were fed a saturated fat-enriched diet for 3 months to determine effects on atherosclerosis. Deletion of AT1a receptors in either endothelial or smooth muscle cells had no discernible effect on the size of atherosclerotic lesions. We also determined the effect of cell-specific AT1a receptor deficiency on atherosclerosis and AAAs using male mice fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min). Again, deletion of AT1a receptors in either endothelial or smooth muscle cells had no discernible effects on either AngII-induced atherosclerotic lesions or AAAs.

Conclusions

Although previous studies have demonstrated whole body AT1a receptor deficiency diminishes atherosclerosis and AAAs, depletion of AT1a receptors in either endothelial or smooth muscle cells did not affect either of these vascular pathologies.     

Transgenic mice expressing Na+-K+-ATPase in smooth muscle decreases blood pressure

The Na(+)-K(+)-ATPase (NKA) is a transmembrane protein that sets and maintains the electrochemical gradient by extruding three Na(+) in exchange for two K(+). An important physiological role proposed for vascular smooth muscle NKA is the regulation of blood pressure via modulation of vascular smooth muscle contractility (5). To investigate the relations between the level of NKA in smooth muscle and blood pressure, we developed mice carrying a transgene for either the NKA alpha(1)- or alpha(2)-isoform (alpha(1 sm+) or alpha(2 sm+) mice) driven by the smooth muscle-specific alpha-actin promoter SMP8. Interestingly, both alpha-isoforms, the one contained in the transgene and the one not contained, were increased to a similar degree at both protein and mRNA levels. The total alpha-isoform protein was increased from 1.5-fold (alpha(1 sm+) mice) to 7-fold (alpha(2 sm+) mice). The increase in total NKA alpha-isoform protein was accompanied by a 2.5-fold increase in NKA activity in alpha(2 sm+) gastric antrum. Immunocytochemistry of the alpha(1)- and alpha(2)-isoforms in alpha(2 sm+) aortic smooth muscle cells indicated that alpha-isoform distributions were similar to those shown in wild-type cells. alpha(2 sm+) Mice (high expression) were hypotensive (109.9 +/- 1.6 vs. 121.3 +/- 1.4 mmHg; n = 13 and 11, respectively), whereas alpha(1 sm+) mice (low expression) were normotensive (122.7 +/- 2.5 vs. 117.4 +/- 2.3; n = 11 or 12). alpha(2 sm+) Aorta, but not alpha(1 sm+) aorta, relaxed faster from a KCl-induced contraction than wild-type aorta. Our results show that smooth muscle displays unique coordinate expression of the alpha-isoforms. Increasing smooth muscle NKA decreases blood pressure and is dependent on the degree of increased alpha-isoform expression.     

Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly

New blood vessels are initially formed through the assembly or sprouting of endothelial cells, but the recruitment of supporting pericytes and vascular smooth muscle cells (mural cells) ensures the formation of a mature and stable vascular network. Defective mural-cell coverage is associated with the poorly organized and leaky vasculature seen in tumors or other human diseases. Here we report that mural cells require ephrin-B2, a ligand for Eph receptor tyrosine kinases, for normal association with small-diameter blood vessels (microvessels). Tissue-specific mutant mice display perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries. Cultured ephrin-B2-deficient smooth muscle cells are defective in spreading, focal-adhesion formation, and polarized migration and show increased motility. Our results indicate that the role of ephrin-B2 and EphB receptors in these processes involves Crk-p130(CAS) signaling and suggest that ephrin-B2 has some cell-cell-contact-independent functions.     

Mesodermal expression of integrin α5β1 regulates neural crest development and cardiovascular morphogenesis

Integrin α5-null embryos die in mid-gestation from severe defects in cardiovascular morphogenesis, which stem from defective development of the neural crest, heart and vasculature. To investigate the role of integrin α5β1 in cardiovascular development, we used the Mesp1^Cre knock-in strain of mice to ablate integrin α5 in the anterior mesoderm, which gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo. Surprisingly, we found that mutant embryos displayed numerous defects related to the abnormal development of the neural crest such as cleft palate, ventricular septal defect, abnormal development of hypoglossal nerves, and defective remodeling of the aortic arch arteries. We found that defects in arch artery remodeling stem from the role of mesodermal integrin α5β1 in neural crest proliferation and differentiation into vascular smooth muscle cells, while proliferation of pharyngeal mesoderm and differentiation of mesodermal derivatives into vascular smooth muscle cells was not defective. Taken together our studies demonstrate a requisite role for mesodermal integrin α5β1 in signaling between the mesoderm and the neural crest, thereby regulating neural crest-dependent morphogenesis of essential embryonic structures.     

MicroRNA-processing enzyme Dicer is required in epicardium for coronary vasculature development

The epicardium is a sheet of epithelial cells covering the heart during early cardiac development. In recent years, the epicardium has been identified as an important contributor to cardiovascular development, and epicardium-derived cells have the potential to differentiate into multiple cardiac cell lineages. Some epicardium-derived cells that undergo epithelial-to-mesenchymal transition and delaminate from the surface of the developing heart subsequently invade the myocardium and differentiate into vascular smooth muscle of the developing coronary vasculature. MicroRNAs (miRNAs) have been implicated broadly in tissue patterning and development, including in the heart, but a role in epicardium is unknown. To examine the role of miRNAs during epicardial development, we conditionally deleted the miRNA-processing enzyme Dicer in the proepicardium using Gata5-Cre mice. Epicardial Dicer mutant mice are born in expected Mendelian ratios but die immediately after birth with profound cardiac defects, including impaired coronary vessel development. We found that loss of Dicer leads to impaired epicardial epithelial-to-mesenchymal transition and a reduction in epicardial cell proliferation and differentiation into coronary smooth muscle cells. These results demonstrate a critical role for Dicer, and by implication miRNAs, in murine epicardial development.     

Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse

Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx1(+/-) mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Here, we identify enhancer elements in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. We describe the generation of transgenic mice that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. We use these Tbx1-Cre mice to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which are derived from Tbx1-expressing cells. We test the hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery.     

Uhrf2 is important for DNA damage response in vascular smooth muscle cells

Emerging evidence shows that Uhrf1 plays an important role in DNA damage response for maintaining genomic stability. Interestingly, Uhrf1 has a paralog Uhrf2 in mammals. Uhrf1 and Uhrf2 share similar domain architectures. However, the role of Uhrf2 in DNA damage response has not been studied yet. During the analysis of the expression level of Uhrf2 in different tissues, we found that Uhrf2 is highly expressed in aorta and aortic vascular smooth muscle cells. Thus, we studied the role of Uhrf2 in DNA damage response in aortic vascular smooth muscle cells. Using laser microirradiation, we found that like Uhrf1, Uhrf2 was recruited to the sites of DNA damage. We dissected the functional domains of Uhrf2 and found that the TTD, PHD and SRA domains are important for the relocation of Uhrf2 to the sites of DNA damage. Moreover, depletion of Uhrf2 suppressed DNA damage-induced H2AX phosphorylation and DNA damage repair. Taken together, our results demonstrate the function of Uhrf2 in DNA damage response.     

Roles of ChlR1 DNA helicase in replication recovery from DNA damage

The ChlR1 DNA helicase is mutated in Warsaw breakage syndrome characterized by developmental anomalies, chromosomal breakage, and sister chromatid cohesion defects. However, the mechanism by which ChlR1 preserves genomic integrity is largely unknown. Here, we describe the roles of ChlR1 in DNA replication recovery. We show that ChlR1 depletion renders human cells highly sensitive to cisplatin; an interstrand-crosslinking agent that causes stalled replication forks. ChlR1 depletion also causes accumulation of DNA damage in response to cisplatin, leading to a significant delay in resolution of DNA damage. We also report that ChlR1-depleted cells display defects in the repair of double-strand breaks induced by the I-PpoI endonuclease and bleomycin. Furthermore, we demonstrate that ChlR1-depeleted cells show significant delays in replication recovery after cisplatin treatment. Taken together, our results indicate that ChlR1 plays an important role in efficient DNA repair during DNA replication, which may facilitate efficient establishment of sister chromatid cohesion.     

p66(ShcA) and oxidative stress modulate myogenic differentiation and skeletal muscle regeneration after hind limb ischemia

Oxidative stress plays a pivotal role in ischemic injury, and p66(ShcA)ko mice exhibit both lower oxidative stress and decreased tissue damage following hind limb ischemia. Thus, it was investigated whether tissue regeneration following acute hind limb ischemia was altered in p66(ShcA)ko mice. Upon femoral artery dissection, muscle regeneration started earlier and was completed faster than in wild-type (WT) control. Moreover, faster regeneration was associated with decreased oxidative stress. Unlike ischemia, cardiotoxin injury induced similar skeletal muscle damage in both genotypes. However, p66(ShcA)ko mice regenerated faster, in agreement with the regenerative advantage upon ischemia. Since no difference between p66(ShcA)wt and knock-out (ko) mice was found in blood perfusion recovery after ischemia, satellite cells (SCs), a resident population of myogenic progenitors, were examined. Similar SCs numbers were present in WT and ko mice. However, in vitro cultured p66(ShcA)ko SCs displayed lower oxidative stress levels and higher proliferation rate and differentiated faster than WT. Furthermore, when exposed to sublethal H(2)O(2) doses, p66(ShcA)ko SCs were resistant to H(2)O(2)-induced inhibition of differentiation. Finally, myogenic conversion induced by MyoD overexpression was more efficient in p66(ShcA)ko fibroblasts compared with WT. The present work demonstrates that oxidative stress and p66(ShcA) play a crucial role in the regenerative pathways activated by acute ischemia.     

V gamma 1+ T cells suppress and V gamma 4+ T cells promote susceptibility to coxsackievirus B3-induced myocarditis in mice

Coxsackievirus B3 infections of C57BL/6 mice, which express the MHC class II IA but not IE Ag, results in virus replication in the heart but minimal myocarditis. In contrast, Bl.Tg.Ealpha mice, which are C57BL/6 mice transgenically induced to express IE Ag, develop significant myocarditis upon Coxsackievirus B3 infection. Despite this difference in inflammatory damage, cardiac virus titers are similar between C57BL/6 and Bl.Tg.Ealpha mice. Removing gammadelta T cells from either strain by genetic manipulation (gammadelta knockout(ko)) changes the disease phenotype. C57BL/6 gammadelta ko mice show increased myocarditis. In contrast, Bl.Tg.Ealpha gammadelta ko mice show decreased cardiac inflammation. Flow cytometry revealed a difference in the gammadelta cell subsets in the two strains, with Vgamma1 dominating in C57BL/6 mice, and Vgamma4 predominating Bl.Tg.Ealpha mice. This suggests that these two Vgamma-defined subsets might have different functions. To test this possibility, we used mAb injection to deplete each subset. Mice depleted of Vgamma1 cells showed enhanced myocarditis, whereas those depleted of Vgamma4 cells suppressed myocarditis. Adoptively transfusing enriched Vgamma4(+) cells to the C57BL/6 and Bl.Tg. Ealpha gammadelta ko strains confirmed that the Vgamma4 subset promoted myocarditis. Th subset analysis suggests that Vgamma1(+) cells biased the CD4(+) T cells to a dominant Th2 cell response, whereas Vgamma4(+) cells biased CD4(+) T cells toward a dominant Th1 cell response.     

Prdm6 Is Essential for Cardiovascular Development In Vivo

Members of the PRDM protein family have been shown to play important roles during embryonic development. Previous in vitro and in situ analyses indicated a function of Prdm6 in cells of the vascular system. To reveal physiological functions of Prdm6, we generated conditional Prdm6-deficient mice. Complete deletion of Prdm6 results in embryonic lethality due to cardiovascular defects associated with aberrations in vascular patterning. However, smooth muscle cells could be regularly differentiated from Prdm6-deficient embryonic stem cells and vascular smooth muscle cells were present and proliferated normally in Prdm6-deficient embryos. Conditional deletion of Prdm6 in the smooth muscle cell lineage using a SM22-Cre driver line resulted in perinatal lethality due to hemorrhage in the lungs. We thus identified Prdm6 as a factor that is essential for the physiological control of cardiovascular development.     

NADPH oxidase but not myeloperoxidase protects lymphopenic mice from spontaneous infections

The adaptive immune system plays an important role in host defense against invading micro-organisms. Yet, mice deficient in T- and B-cells are surprisingly healthy and develop few spontaneous infections when raised under specific pathogen-free conditions (SPF). The objective of this study was to ascertain what role phagocyte-associated NADPH oxidase or myeloperoxidase (MPO) plays in host defense in mice lacking both T- and B-cells. To do this, we generated lymphopenic mice deficient in either NADPH oxidase or MPO by crossing gp91(phox)-deficient (gp91 ko) or MPO ko mice with mice deficient in recombinase activating gene-1 (RAG ko). We found that neither gp91 ko, MPO ko mice nor lymphocyte-deficient RAG ko mice developed spontaneous infections when raised under SPF conditions and all mice had life spans similar to wild-type (WT) animals. In contrast, gp91xRAG double-deficient (DKO) but not MPOxRAG DKO mice developed spontaneous multi-organ bacterial and fungal infections early in life and lived only a few months. Infections in the gp91xRAG DKO mice were characterized by granulomatous inflammation of the skin, liver, heart, brain, kidney, and lung. Addition of antibiotics to the drinking water attenuated the spontaneous infections and increased survival of the mice. Oyster glycogen-elicited polymorphonuclear neutrophils (PMNs) and macrophages obtained from gp91 ko and gp91xRAG DKO mice had no detectable NADPH oxidase activity whereas WT, RAG ko, and MPOxRAG DKO PMNs and macrophages produced large and similar amounts of superoxide in response to phorbol myristate acetate. The enhanced mortality of the gp91xRAG DKO mice was not due to defects in inflammatory cell recruitment or NO synthase activity (iNOS) as total numbers of elicited PMNs and macrophages as well as PMN- and macrophage-derived production of nitric oxide-derived metabolites in these mice were similar and not reduced when compared to that of WT mice. Taken together, our data suggest that that NADPH oxidase but not MPO (nor iNOS) is required for host defense in lymphopenic mice and that lymphocytes and NADPH oxidase may compensate for each other's deficiency in providing resistance to spontaneous bacterial infections.     

Defective paracrine signalling by TGFbeta in yolk sac vasculature of endoglin mutant mice: a paradigm for hereditary haemorrhagic telangiectasia

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder in humans that is characterised by multisystemic vascular dyplasia and recurrent haemorrhage. Germline mutations in one of two different genes, endoglin or ALK1 can cause HHT. Both are members of the transforming growth factor (TGF) beta receptor family of proteins, and are expressed primarily on the surface of endothelial cells (ECs). Mice that lack endoglin or activin receptor like kinase (ALK) 1 die at mid-gestation as a result of defects in the yolk sac vasculature. Here, we have analyzed TGFbeta signalling in yolk sacs from endoglin knockout mice and from mice with endothelial-specific deletion of the TGFbeta type II receptor (TbetaRII) or ALK5. We show that TGFbeta/ALK5 signalling from endothelial cells to adjacent mesothelial cells is defective in these mice, as evidenced by reduced phosphorylation of Smad2. This results in the failure of vascular smooth muscle cells to differentiate and associate with endothelial cells so that blood vessels remain fragile and become dilated. Phosphorylation of Smad2 and differentiation of smooth muscle can be rescued by culture of the yolk sac with exogenous TGFbeta1. Our data show that disruption of TGFbeta signalling in vascular endothelial cells results in reduced availability of TGFbeta1 protein to promote recruitment and differentiation of smooth muscle cells, and provide a possible explanation for weak vessel walls associated with HHT.     

Genetic Evidence for XPC-KRAS Interactions During Lung Cancer Development

Lung cancer causes more deaths than breast, colorectal and prostate cancers combined. Despite major advances in targeted therapy in a subset of lung adenocarcinomas, the overall 5-year survival rate for lung cancer worldwide has not significantly changed for the last few decades. DNA repair deficiency is known to contribute to lung cancer development. In fact, human polymorphisms in DNA repair genes such as xeroderma pigmentosum group C(XPC) are highly associated with lung cancer incidence. However, the direct genetic evidence for the role of XPC for lung cancer development is still lacking. Mutations of the Kirsten rat sarcoma viral oncogene homolog(Kras) or its downstream effector genes occur in almost all lung cancer cells, and there are a number of mouse models for lung cancer with these mutations. Using activated Kras, Kras LA1, as a driver for lung cancer development in mice, we showed for the first time that mice with Kras LA1 and Xpc knockout had worst outcomes in lung cancer development, and this phenotype was associated with accumulated DNA damage. Using cultured cells, we demonstrated that induced expression of oncogenic KRAS G12 V led to increased levels of reactive oxygen species(ROS) as well as DNA damage, and both can be suppressed by anti-oxidants. Our results suggest that XPC may help repair DNA damage caused by KRAS-mediated production of ROS.     

Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions.

Hyperglycemia-induced oxidative stress may play a key role in the pathogenesis of diabetic vascular disease. The purpose of this study was to determine the effects of glucose on levels of glutathione (a major intracellular antioxidant), the expression of gamma-glutamylcysteine synthetase (the rate-limiting enzyme in glutathione de novo synthesis), and DNA damage in human vascular smooth muscle cells in vitro. High glucose conditions and buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase, reduced intracellular glutathione levels in vascular smooth muscle cells. This reduction was accompanied by a decrease in the mRNA expression of both subunits of gamma-glutamylcysteine synthetase as well as an increase in DNA damage. In high glucose conditions, incubation of the vascular smooth muscle cells with alpha-lipoic acid and L-cystine restored glutathione levels. We suggest that the decrease in GSH levels seen in high glucose conditions is mediated by the availability of cysteine (rate-limiting substrate in de novo glutathione synthesis) and the gene expression of the gamma-glutamylcysteine synthetase enzyme. Glutathione depletion is associated with an increase in DNA damage, which can be reduced when glutathione levels are restored.