Vicente Andrés

MYOD-INDUCED EXPRESSION OF P21 INHIBITS CYCLIN-DEPENDENT KINASE ACTIVITY UPON MYOCYTE TERMINAL DIFFERENTIATION

The terminal differentiation of C2C12 skeletal muscle cells involves the activation of unique sets of genes and an irreversible withdrawal from the cell cycle. This process is associated with a decrease in cdk2 activity in cell extracts. The decrease in cdk2 activity correlates with diminished levels of cdk2 and cyclin A and with a marked induction of the p21 cyclin-dependent kinase (cdk) inhibitor. The upregulation of p21 occurred at the levels of mRNA and protein, and p21 formed a complex with the cyclin kinases in myotubes. Further, the immunodepletion of p21 from myotube extracts neutralized the heat-stable cdk2 inhibitory activity that was induced upon myogenic differentiation. The levels of p21 mRNA, protein, and activity remained constant in myotubes when they were reexposed to mitogen-rich growth medium, indicating that permanent changes in the cells genetic program contribute to its sustained expression following terminal differentiation. Indeed, 10T1/2 fibroblasts transformed with the myogenic factor MyoD, but not the parental multipotent cells, upregulated p21 transcript levels when induced to differentiate by serum withdrawal, demonstrating that the upregulation is an integral feature of myogenic commitment and differentiation. The functional consequences of this upregulation were indicated by ectopically expressing p21 in myoblasts; this was sufficient for cell cycle arrest in mitogen-rich growth medium. The induction and sustained expression of p21 appears to be a contributory mechanism by which myocytes irreversibly exit the cell cycle upon terminal differentiation.

Histopathology of In-Stent Restenosis in Patients With Peripheral Artery Disease

BACKGROUND Clinical studies have suggested that smooth muscle cell (SMC) hyperplasia is the most likely cause of in-stent restenosis. However, pathological data regarding this issue are limited. Specifically, direct evidence of proliferative activity in tissues excised from stenotic stents has not been previously reported. METHODS AND RESULTS Tissue specimens were retrieved by directional atherectomy from 10 patients in whom in-stent restenosis complicated percutaneous revascularization of peripheral artery disease. Analysis of cellular composition was performed quantitatively after cell-specific immunostaining. For specimens preserved in methanol (7 of 10), cellular proliferation was evaluated by use of antibodies to proliferating cell nuclear antigen (PCNA), cyclin E, and cdk2. TUNEL staining for apoptosis was performed on 8 paraformaldehyde-preserved specimens. Each of the 10 specimens contained extensive foci of hypercellularity composed predominantly of SMCs (mean+/-SEM, 59.3+/-3.0%). Evidence of ongoing proliferative activity was documented in all 7 methanol-preserved specimens: 24.6+/-2.3% of SMCs were PCNA-positive, 24.8+/-3.1% were cyclin E-positive, and 22.5+/-2.2% were cdk2-positive. Apoptotic cells were detected in all 8 specimens that had been appropriately preserved to permit DNA nick-end labeling. Macrophages and leukocytes were identified in each of the 10 specimens but accounted for a proportionately smaller number of cells (14.5+/-1.9% and 9.5+/-1.4%, respectively). Organized thrombus was observed in 6 of the 10 specimens. CONCLUSIONS These findings support the notion that in-stent restenosis results from SMC hyperplasia and suggest that adjunctive therapies designed to inhibit SMC proliferation may further enhance the utility of endovascular stents.

Role of A-type lamins in signaling, transcription, and chromatin organization

A-type lamins (lamins A and C), encoded by the LMNA gene, are major protein constituents of the mammalian nuclear lamina, a complex structure that acts as a scaffold for protein complexes that regulate nuclear structure and functions. Interest in these proteins has increased in recent years with the discovery that LMNA mutations cause a variety of human diseases termed laminopathies, including progeroid syndromes and disorders that primarily affect striated muscle, adipose, bone, and neuronal tissues. In this review, we discuss recent research supporting the concept that lamin A/C and associated nuclear envelope proteins regulate gene expression in health and disease through interplay with signal transduction pathways, transcription factors, and chromatin-associated proteins.

Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27(KIP1), an inhibitor of neointima formation in the rat carotid artery.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia during atherosclerosis and restenosis, but the endogenous cell cycle regulatory factors underlying VSMC growth in response to arterial injury are not well understood. In the present study, we report that downregulation of cyclin-dependent kinase 2 (cdk2) activity in serum-deprived VSMCs was associated with the formation of complexes between cdk2 and its inhibitory protein p27(KIP1) (p27). Ectopic overexpression of p27 in serum-stimulated VSMCs resulted in the inhibition of cdk2 activity and repression of cyclin A promoter activity. Collectively, these findings indicate that p27 may contribute to VSMC growth arrest in vitro. Using the rat carotid model of balloon angioplasty, a marked upregulation of p27 was observed in injured arteries. High levels of p27 expression in the media and neointima correlated with downregulation of cdk2 activity at 2 wk after angioplasty, and adenovirus-mediated overexpression of p27 in balloon-injured arteries attenuated neointimal lesion formation. Thus, the inhibition of cdk2 function and repression of cyclin A gene transcription through the induction of the endogenous p27 protein provides a mechanism for the inhibition of VSMC growth at late time points after angioplasty.

Telomeres and Cardiovascular Disease: Does Size Matter?

Telomeres-the specialized DNA-protein structures at the ends of eukaryotic chromosomes-are essential for maintaining genome stability and integrity and for extended proliferative life span in both cultured cells and in the whole organism. Telomerase and additional telomere-associated proteins are necessary for preserving telomeric DNA length. Age-dependent telomere shortening in most somatic cells, including vascular endothelial cells, smooth muscle cells, and cardiomyocytes, is thought to impair cellular function and viability of the aged organism. Telomere dysfunction is emerging as an important factor in the pathogenesis of hypertension, atherosclerosis, and heart failure. In this Review, we discuss present studies on telomeres and telomere-associated proteins in cardiovascular pathobiology and their implications for therapeutics.

Splicing-Directed Therapy in a New Mouse Model of Human Accelerated Aging

Antisense oligonucleotides reverse premature aging and extend life span in mutant mice that mimic aberrant splicing in progeria patients. Countering Careless Cutting Carpenters warn that one should “measure twice, cut once” to avoid unfixable assaults on building materials. Indeed, careless cutting lies at the heart of Hutchinson-Gilford progeria syndrome (HGPS). This premature aging disease is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site in LMNA RNA; aberrant cutting and splicing results in the production of an mRNA that encodes progerin, a truncated form of the lamin A protein that is also produced in small amounts during normal aging. Until now, no model system has recapitulated the pathogenic LMNA splicing that occurs in HGPS patients. Here, Osorio et al. characterize such HGPS mutant mice mimics—called LmnaG609G/G609G mice—and show that antisense oligonucleotide–based therapy reverses various premature aging phenotypes and extends life span. Encoded by the LMNA gene, lamin A is a nuclear envelope protein that is important for nuclear stability, chromatin structure, and regulation of gene expression. Osorio et al. showed that the LmnaG609G/G609G mice produced reduced amounts of intact lamin A, accumulated progerin, displayed the nuclear abnormalities and transcriptional alterations seen in other progeroid models, and sported the key clinical features of human HGPS, such as a shortened life span, reduced size, disrupted metabolism, and enhanced bone and cardiovascular maladies relative to wild-type animals. The authors then used their newly characterized HGPS animal model to test the effects of antisense morpholino oligonucleotides that bound to and blocked the aberrant splice donor site in Lmna RNA. These reagents reduced progerin accumulation and corrected the nuclear abnormalities in both cultured mutant mouse and human HGPS fibroblasts. Furthermore, LmnaG609G/G609G mice that were treated with a combination of two antisense oligonucleotides that blocked aberrant splicing displayed reduced amounts of accumulated progerin, enhanced life expectancy, and a reversal of the phenotypical and molecular alterations associated with HGPS, including the righting of gene expression aberrations and normalization of blood glucose levels. Together, these findings provide preclinical proof of concept for the use of antisense oligonucleotide–based therapies in the treatment of HGPS. Furthermore, because progerin also accumulates during normal aging, the LmnaG609G/G609G mutant mice may be useful for preclinical testing of therapies designed to slow the human aging process and prevent age-related diseases. As the poet Ralph Waldo Emerson noted, “All diseases run into one—old age.” Hutchinson-Gilford progeria syndrome (HGPS) is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site and yields a truncated form of prelamin A called progerin. Small amounts of progerin are also produced during normal aging. Studies with mouse models of HGPS have allowed the recent development of the first therapeutic approaches for this disease. However, none of these earlier works have addressed the aberrant and pathogenic LMNA splicing observed in HGPS patients because of the lack of an appropriate mouse model. Here, we report a genetically modified mouse strain that carries the HGPS mutation. These mice accumulate progerin, present histological and transcriptional alterations characteristic of progeroid models, and phenocopy the main clinical manifestations of human HGPS, including shortened life span and bone and cardiovascular aberrations. Using this animal model, we have developed an antisense morpholino–based therapy that prevents the pathogenic Lmna splicing, markedly reducing the accumulation of progerin and its associated nuclear defects. Treatment of mutant mice with these morpholinos led to a marked amelioration of their progeroid phenotype and substantially extended their life span, supporting the effectiveness of antisense oligonucleotide–based therapies for treating human diseases of accelerated aging.

Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope

Sequestration of c-Fos at the nuclear envelope (NE) through interaction with A-type lamins suppresses AP-1–dependent transcription. We show here that c-Fos accumulation within the extraction-resistant nuclear fraction (ERNF) and its interaction with lamin A are reduced and enhanced by gain-of and loss-of ERK1/2 activity, respectively. Moreover, hindering ERK1/2-dependent phosphorylation of c-Fos attenuates its release from the ERNF induced by serum and promotes its interaction with lamin A. Accordingly, serum stimulation rapidly releases preexisting c-Fos from the NE via ERK1/2-dependent phosphorylation, leading to a fast activation of AP-1 before de novo c-Fos synthesis. Moreover, lamin A–null cells exhibit increased AP-1 activity and reduced levels of c-Fos phosphorylation. We also find that active ERK1/2 interacts with lamin A and colocalizes with c-Fos and A-type lamins at the NE. Thus, NE-bound ERK1/2 functions as a molecular switch for rapid mitogen-dependent AP-1 activation through phosphorylation-induced release of preexisting c-Fos from its inhibitory interaction with lamin A/C.

Determination of the Consensus Binding Site for MEF2 Expressed in Muscle and Brain Reveals Tissue-specific Sequence Constraints

The myocyte-specific enhancer factor-2 (MEF2) proteins are expressed in the three major types of muscle (skeletal, cardiac, and smooth) and function as transcriptional activators of muscle-specific and growth factor-regulated genes through binding to a canonical A/T-rich cis-element. Although MEF2 proteins are also expressed in brain, MEF2-regulated muscle-specific gene products are not detected in this tissue. To gain insight into the regulation of MEF2 function in vivo, we have selected its optimal DNA targets from a library of degenerate oligonucleotides using anti-MEF2A antibodies and cell extracts from skeletal muscle, heart, and brain. The consensus binding site in these three tissues contains an indistinguishable core motif, 5′-CT(A/t)(a/t)AAATAG-3′. However, the optimal target for MEF2 expressed in the brain shows additional sequence constraints (5′-TGTTACT(A/t)(a/t)AAATAGA(A/t)-3′) that are not observed in the sequences selected with skeletal and cardiac muscle extracts. Thus, differences in DNA binding preferences of MEF2 proteins in muscle and brain may contribute to tissue-specific gene expression during myogenesis and neurogenesis.

Clonal hematopoiesis associated with Tet2 deficiency accelerates atherosclerosis development in mice

Faulty blood cells and heart disease Recent studies have shown that elderly peoples blood cells often harbor mutations in genes encoding certain epigenetic regulators. These mutations can lead to clonal expansion of the mutant blood cells, which increases the risk of blood cancers and cardiovascular disease. Fuster et al. generated a mouse model to investigate how one of these genes, Tet2, affects atherosclerosis development (see the Perspective by Zhu et al.). They found that the disease progressed more rapidly in mice transplanted with Tet2-deficient bone marrow cells. This was due to increased secretion of interleukin-1β by Tet2-deficient macrophages in a process that depended on the action of inflammasomes. Science, this issue p. 842; see also p. 798 Bone marrow deficient in a gene frequently mutated in blood cells of elderly humans promotes atherosclerosis in mice. Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of Tet2-mutant cells in atherosclerosis-prone, low-density lipoprotein receptor–deficient (Ldlr–/–) mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome–mediated interleukin-1β secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic TET2 mutations in blood cells play a causal role in atherosclerosis.

Antibody Blockade of Thrombospondin Accelerates Reendothelialization and Reduces Neointima Formation in Balloon-Injured Rat Carotid Artery

BACKGROUND Remodeling of the extracellular matrix plays an important role during the pathogenesis of atherosclerosis and restenosis. The matrix glycoprotein thrombospondin-1 (TSP1) inhibits endothelial cell proliferation and migration in vitro. In contrast, TSP1 facilitates the growth and migration of cultured vascular smooth muscle cells. Accordingly, we investigated the hypothesis that administration of anti-TSP1 antibody could facilitate reendothelialization and inhibit neointimal thickening in balloon-injured rat carotid artery. METHODS AND RESULTS Sprague-Dawley rats were subjected to left common carotid artery denudation, after which arteries were treated with C6.7 anti-TSP1 or control antibody. Evans blue dye staining 2 weeks after injury disclosed significantly increased reendothelialization in arteries treated with C6.7 antibody compared with the control group, and this effect was associated with increased number of proliferating cell nuclear antigen-positive endothelial cells. In contrast, treatment with C6.7 antibody decreased the number of proliferating cell nuclear antigen-positive vascular smooth muscle cells in the injured arterial wall. Neointimal thickening was correspondingly attenuated to a statistically significant degree in arteries receiving C6.7 antibody versus the control group at both the 2-week and 4-week time points. CONCLUSIONS Intra-arterial delivery of antibody against TSP1 facilitated reendothelialization and reduced neointimal lesion formation after balloon denudation.