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R T Allen

Publications and source records attributed to R T Allen.

10 recordsLinked to original sources
PubMed2006

Characterization of pro-apoptotic and matrix-degradative gene expression following induction of osteoarthritis in mature and aged rabbits.

OBJECTIVE: The genetic and molecular changes leading to the distinctive alterations of aged cartilage and its propensity for developing osteoarthritis (OA) are unknown. We hypothesized that pro-apoptotic and matrix-degradative gene expression in a rabbit model of induced OA using mature and aged animals might elucidate this relationship. METHODS: Groups of six mature and aged rabbits underwent anterior cruciate ligament transection (ACLT) and were sacrificed 4 weeks after surgery to create an Outerbridge grade II OA. RNA was extracted from the articular cartilage and menisci of the affected knee and was examined with regard to expression of the following genes: Caspase 8, Fas, Fas ligand (Fas-L), p53, aggrecanase, matrix metalloproteinase (MMP)-1, and MMP-3-MMP-13. A second cohort of mature and aged animals was sacrificed with no intervention to the joint and gene expression was assessed in a similar manner. RESULTS: Fas and Caspase 8 showed significantly increased expression in the cartilage of mature animals with induced OA when compared to unoperated controls while induction of OA in aged rabbits did not significantly increase expression of any of the apoptosis genes. Among unoperated animals, the aged cohort showed significantly increased expression of MMP-1 and aggrecanase in cartilage when compared to mature animals. MMP-13 expression was upregulated in aged cartilage following induction of OA. Although ACLT animals showed gross thinning and irregularities within the meniscus, only the expression of Caspase 8 in the aged rabbits was significantly increased after induction of OA. CONCLUSIONS: Aging of articular cartilage shares some qualities with the development of OA, as seen in the parallel increases in gene expression of Caspase 8 and Fas. Although this may imply a common mechanism of cartilage degeneration in aging and OA or even a spectrum of disease, both are complex processes requiring further study.

Aging↗
PubMed2005

Sustained Akt/PKB activation and transient attenuation of c-jun N-terminal kinase in the inhibition of apoptosis by IGF-1 in vascular smooth muscle cells.

Characteristics of hVSMC apoptosis and its inhibition by insulin-like growth factor-1 (IGF-1) remain unclear. Also unclear is whether a balance in hVSMCs exists whereby c-Jun N-terminal stress kinases (JNK) promote apoptosis while extracellular signal-regulated (ERK1/2) MAP kinases inhibit cell death. In this study, we examined the involvement of Akt/PKB and its upstream kinase, PDK1 and whether JNK activation correlated with human and rat VSMC apoptosis induced by staurosporine and by c-myc, respectively. We observed a strong, sustained JNK activation (and c-Jun phosphorylation), which correlated with VSMC apoptosis. IGF-1 (13.3 nM), during apoptosis inhibition, transiently inhibited JNK activity at 1 h in a phosphatidylinositol 3-kinase (PI3-K)- and MEK-ERK-dependent manner, as wortmannin (100 nM) or PD98059 (30 muM) partially attenuated the IGF-1 effect. PKC down-regulation had no effect on JNK inhibition by IGF-1. While IGF-1 alone produced a strong phosphorylation of Akt/PKB in hVSMCs up to 6 h, it was notably stronger and more sustained during ratmyc and hVSMCs apoptosis inhibition. Further, whereas transient expression of phosphorylated Akt protected VSMCs from apoptosis by nearly 50%, expression of dominant interfering alleles of Akt or PDK1 strongly inhibited IGF-1-mediated VSMC survival. These results demonstrate for the first time that transient inhibition of a pro-apoptotic stimulus in VSMCs may be sufficient to inhibit a programmed cell death and that sustained anti-apoptotic signals (Akt) elicited by IGF-1 are augmented during a death stimulus. Furthermore, PI3-K and ERK-MAPK pathways may cooperate to protect VSMCs from cell death.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1998

Morphologic and temporal analysis of vascular smooth muscle cell apoptosis induced by c-myc and E1A.

Apoptosis is a physiologic form of cell death present in many disease conditions. When the balance of mitosis versus apoptosis is altered, tumor-like growth or degeneration of tissues may ensue. This appears to occur in several diseases, including those of the cardiovascular system, where apoptosis plays a key role in atherosclerosis and restenosis following angioplasty. Since c-myc is upregulated in the pathogenesis of these diseases, we chose to study the sequential morphologic features of programmed cell death in vascular smooth muscle cells induced by c-myc and by the adenovirus early gene E1A. Morphology and timed events in apoptotic cell cultures were analyzed by scanning electron microscopy, transmission electron microscopy, and time-lapse videomicroscopy. We observed that both c-myc- and E1A-induced apoptosis (in serum-free medium) resulted in numerous, tightly packed clusters of apoptotic blebs, as well as in one or two asymmetrically larger blebs. Transmission electron miscroscopy analysis revealed the larger blebs contained mostly nuclear chromatin, whereas the many smaller fragments often had little or no chromatin. Time-lapse studies showed that apoptosis was induced at a slower rate in cells stably transfected with c-myc versus those stably transfected with E1A. The early changes of apoptosis, including cell shrinkage and intense blebbing, occurred in under 5 min in both cells. Slight alterations such as cell size and further rounding occurred up to 8 h following the initial changes of apoptosis. Rather than being a part of the apoptotic response, release from the culture floor almost entirely resulted from movement of the culture flask. These studies provide a framework of timed morphologic events for future mechanistic investigation into the key aspects of myc- and E1A-induced apoptosis in vascular smooth muscle.

Adenovirus E1A Proteins↗
PubMed1998

Mechanisms controlling cellular suicide: role of Bcl-2 and caspases.

Apoptosis is an essential and highly conserved mode of cell death that is important for normal development, host defense and suppression of oncogenesis. Faulty regulation of apoptosis has been implicated in degenerative conditions, vascular diseases, AIDS and cancer. Among the numerous proteins and genes involved, members of the Bcl-2 family play a central role to inhibit or promote apoptosis. In this article, we present up-to-date information and recent discoveries regarding biochemical functions of Bcl-2 family proteins, positive and negative interactions between these proteins, and their modification and regulation by either proteolytic cleavage or by cytosolic kinases, such as Raf-1 and stress-activated protein kinases. We have critically reviewed the functional role of caspases and the consequences of cleaving key substrates, including lamins, poly(ADP ribose) polymerase and the Rb protein. In addition, we have presented the latest Fas-induced signalling mechanism as a model for receptor-linked caspase regulation. Finally, the structural and functional interactions of Ced-4 and its partial mammalian homologue, apoptosis protease activating factor-1 (Apaf-1), are presented in a model which includes other Apafs. This model culminates in a caspase/Apaf regulatory cascade to activate the executioners of programmed cell death following cytochrome c release from the mitochondria of mammalian cells. The importance of these pathways in the treatment of disease is highly dependent on further characterization of genes and other regulatory molecules in mammals.

Animals↗
PubMed1997

Cell adhesion molecules and insulin-like growth factor-1 in vascular disease.

PURPOSE: Recent advances in the understanding of the biologic mechanisms of vascular diseases suggest that multifactorial stimulation of the endothelial cell and its subsequent adhesion to leukocytes is a prerequisite to the formation of atherosclerotic and restenotic lesions. As leukocyte-endothelial cell interaction is coordinated by a variety of cell adhesion molecules (CAMs), we hypothesized that the expression of certain CAMs is up-regulated in the vasculature of patients who have peripheral vascular disease. In addition, we proposed that insulin-like growth factor-1 (IGF-1) increases monocyte-endothelial adhesion by means of upregulation of these CAMs. METHODS: Using immunohistochemical techniques, the expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin was examined in human vascular disease specimens. Normal aortas obtained from the organ retrieval system were studied as control specimens. Adhesion studies between human umbilical vein endothelial cells (HUVECs) incubated with IGF-1 and purified human blood monocytes labeled with 51chromium were completed. Western blotting and flow cytometry were performed to show CAM expression on IGF-1-treated HUVECs. RESULTS: Of the CAMs, ICAM-1, P-selectin, and E-selectin were distinctly increased in diseased specimens when compared with control specimens (p < 0.05). Adhesion studies showed an increase in monocyte-endothelial cell adhesion of as much as 40% to 45% (p < 0.01) over baseline, with peak adherence occurring 4 hours after treatment with IGF-1. IGF-1 increased adherence in a dose- and time-dependent manner. The threshold concentration of IGF-1 that induced increased adhesion was 20 ng/ml, with a maximum effect occurring at 150 ng/ml. This increased adhesion was attenuated by pretreatment with IGF-I receptor antibody, as well as with genistein and herbimycin-A, which are potent and selective tyrosine kinase inhibitors. Increased adhesion correlated with an increase in the expression of CAMs on the surface of the HUVECs. An additive effect on adhesion was observed between IGF-1 and tumor necrosis factor-alpha (TNF-alpha) and endothelin-1 (ET-1). Finally, immunohistochemical analysis of human vascular disease specimens revealed an increased expression of IGF-1 receptors as compared with control specimens (p < 0.05). CONCLUSIONS: These results suggest that IGF-1 may be important in the pathogenesis of peripheral vascular disease by increasing endothelial cell-monocyte adhesion by means of an increase in the expression of ICAM-1 and VCAM-1.

Aorta↗
PubMed1997

Morphological and biochemical characterization and analysis of apoptosis.

Apoptosis is a form of programmed cell death serving physiologic and homeostatic functions. However, recent evidence implicating apoptosis in the etiology and pathophysiology of known human diseases, such as heart diseases, cancer, AIDS, and neurodegenerative and autoimmune diseases are continually surfacing. This has spawned the need for identifying which methods are the most effective and well accepted to decipher its presence in a variety of research settings. We have therefore detailed the morphology and biochemical features of apoptotic cell death, with an emphasis on discriminating it from necrosis. In addition, we describe specific and selective techniques which are optimal to target hallmark apoptotic features, such as microscopy, Annexin V labeling, in situ nick-end labeling (TUNEL), and DNA fragmentation analysis by gel electrophoresis and ELISA for oligonucleosome-sized DNA. The advantages and disadvantages of each technique are discussed, as well as their experimental importance relative to one another. The methods have been described in a stepwise fashion, and can readily be applied in the majority of cell systems. Whether working on the tissue or single cell level, these methods are highly effective in qualifying and quantifying apoptosis. The application of these methods in conjunction with molecular techniques can further delineate the underlying mechanisms of apoptosis.

Acquired Immunodeficiency Syndrome↗