An internal ribosome entry site mediates the initiation of soluble guanylyl cyclase beta2 mRNA translation.

The soluble guanylyl cyclases (sGC), the receptor for nitric oxide, are heterodimers consisting of an alpha- and beta-subunit. This study aimed to investigate the translational mechanism of the sGC beta2-subunit. Two mRNA species for sGC beta2 were isolated from human kidney. These transcripts had dissimilar 5'-untranslated regions (5'-UTRs). The most abundant sGC beta2 mRNA showed numerous upstream open reading frames (ORFs) and stable secondary structures that inhibited in vivo and in vitro translation. To evaluate whether these 5'-UTRs harbored an internal ribosome entry site (IRES) that allows translation by an alternative mechanism, we inserted these regions between the two luciferase genes of a bicistronic vector. Transfection of those genetic constructs into HeLa cells demonstrated that both sGC beta2 leaders had IRES activity in a cell-type dependent manner. Finally, the secondary structural model of the sGC beta2 5'-UTR predicts a Y-type pseudoknot that characterizes the IRES of cellular mRNAs. In conclusion, our findings suggest that sGC beta2 5'-UTRs have IRES activity that may permit sGC beta2 expression under conditions that are not optimal for scanning-dependent translation.

Aging and transplant arteriosclerosis in absence of alloreactivity and immunosuppressive drugs in a rat aortic model: recipient age's contribution.

BACKGROUND: Almost half of all transplanted vascularized organ grafts will be lost to transplant arteriosclerosis sometime posttransplantation. Organ shortage for primary transplants and retransplants has led to donor-pool expansion to include elderly donors, knowing that aging per se promotes arteriosclerosis. The current understanding that donor age negatively affects organ and/or patient survival outcome is undermined by variables such as the use of immunosuppressive drugs, their toxicity to the graft, degree of donor-recipient histocompatibility, and the resulting chronic rejection. The purpose of this study was to determine whether the donor's age or recipient's age matters the most in transplant arteriosclerosis in the absence of such variables. METHODS: A syngeneic combination was used where young (2-month-old) and old (22-month-old) donor aortas were injured to initiate neointimal thickening, then transplanted into age-mismatched recipients for 14, 60, and 90 days and then assessed for neointimal thickening. Base level injury response due ischemia and surgery was evaluated in age-matched and noninjured aortic grafts, respectively. RESULTS: Young aortas invariably developed thicker neointima when transplanted into old recipients than when transplanted into young ones. Correspondingly, old aortas transplanted in young recipients consistently developed less neointimal thickening than when transplanted into old recipients. CONCLUSIONS: Our findings strongly suggest that the severity of age-related neointima formation is primarily determined by the recipient's age rather than the donor's age. Therefore, in addition to focusing on donor-specific tolerance induction, strategies aiming at increasing the lifespan of vascularized organ grafts also have to take into consideration the recipient's aging milieu.

Cryptic endotoxic nature of Bacillus thuringiensis Cry1Ab insecticidal crystal protein.

Cry1Ab is one of the most studied insecticidal proteins produced by Bacillus thuringiensis during sporulation. Structurally, this protoxin has been divided in two domains: the N-terminal toxin core and the C-terminal portion. Although many studies have addressed the biochemical characteristics of the active toxin that corresponds to the N-terminal portion, there are just few reports studying the importance of the C-terminal part of the protoxin. Herein, we show that Cry1Ab protoxin has a unique natural cryptic endotoxic property that is evident when their halves are expressed individually. This toxic effect of the separate protoxin domains was found against its original host B. thuringiensis, as well as to two other bacteria, Escherichia coli and Agrobacterium tumefaciens. Interestingly, either the fusion of the C-terminal portion with the insecticidal domain-III or the whole N-terminal region reduced or neutralized such a toxic effect, while a non-Cry1A peptide such as maltose binding protein did not neutralize the toxic effect. Furthermore, the C-terminal domain, in addition to being essential for crystal formation and solubility, plays a crucial role in neutralizing the toxicity caused by a separate expression of the insecticidal domain much like a dot/anti-dot system.

Long-term acceptance of composite tissue allografts through mixed chimerism and CD28 blockade.

BACKGROUND: Clinical composite-tissue (hand) transplantation between genetically disparate individuals currently requires potent, nonspecific immunosuppressive agents that are neither completely successful in preventing acute episodes of rejection nor free from complications. The reliance on long-term immunosuppression has prompted this study to achieve donor-specific transplantation tolerance in adult recipients using a nontoxic, nonmyeloablative protocol. METHODS: Fully mismatched, 4- to 6-week-old ACI (RT1Aa) and Wistar Furth (WF) rats were used as donors and recipients, respectively. Recipients were administered CTLA4-Ig at 2 mg/kg per day (alternate days) in combination with tacrolimus at 1 mg/kg per day (daily) from day 0 through day +10, antilymphocyte serum at 10 mg at day +10 (single dose), and total-body irradiation t 300 cGy (day 0) before bone-marrow transplantation (BMT) (day 0) with 100 x 10(6) T-cell-depleted bone marrow cells. Hindlimb transplants were performed 4 weeks postBMT. Multilineage donor hematopoiesis was determined pre- and posttransplant using flow cytometry. In vitro T-cell responses were evaluated by mixed lymphocyte reactivity assays. RESULTS: CD28 blockade in a transplant model of mixed chimerism effectively aborts T-cell clonal expansion in vitro and in vivo, inhibits the development of acute and chronic rejection of vascularized hindlimb allografts in rats (ACI limbs to ACI-->WF chimeras, n=5; WF limbs to ACI-->WF chimeras, n=4), and subsequently leads to long-term survival of allogeneic skin grafts (n=9). Third-party (F344, n=4) transplants were uniformly rejected within 14 days posttransplant. Multilineage donor hematopoiesis was demonstrated pre- and posttransplant. Donor chimerism, present postBMT, increased throughout the study (pretransplant range 2-28%, mean 17%; posttransplant range 5-49%, mean 34%). Transplant recipients maintained full reactivity to respond to third-party antigens without harmful manifestations of graft-versus-host disease. CONCLUSIONS: Although efforts have been made to induce tolerance to composite tissue allografts in adult recipients, thus far, none have succeeded without toxic, myeloablative host preconditioning. Our demonstration that tolerance can be achieved with minimal preconditioning provides a rationale for application to large animals and humans and suggests that although composite tissue allografts may have a significant skin component (and are therefore felt to be highly antigenic), protocols used to induce tolerance to organ transplants may be equally applicable to composite-tissue allotransplantation.

Use of recombinase activation gene-2 deficient mice to ascertain the role of cellular and humoral immune responses in the development of chronic rejection.

INTRODUCTION: Given its multifactorial etiology, the relative contribution of anti-donor cellular and humoral immune responses in the pathogenesis of chronic rejection is as yet ambiguous. We hypothesized that alloreactive T and B cells play a seminal role in the development of this lesion. METHODS: To address this hypothesis, RAG-2(-/-) mice were used as donors and recipients in a well-established murine model of aortic transplantation. Grafts were transplanted across the following groups: Group I: C3H --> C3H; Group II: Wild-type [WT] 129Sv (H-2(b)) --> C3H (H-2(k)); Group III: C3H --> WT 129Sv; Group IV: 129SvEv RAG-2(-/-) --> C3H; and Group V: C3H --> 129SvEv RAG-2(-/-). Grafts were harvested at d40 to 146 post-transplantation for morphologic and immunohistochemical analyses and semi-quantitative RT-PCR was employed to evaluate the intragraft mRNA expression of various immune mediators. Mixed lymphocyte reaction and complement-mediated alloantibody cytotoxicity assays were performed to determine anti-donor proliferative and humoral responses, respectively. RESULTS: Unlike that across the syngeneic combination (Group I), marked intimal thickening with corresponding luminal narrowing was observed in the majority of the aortic allografts (Groups II-IV). On the contrary, the morphology of C3H aortic allografts harvested from the majority of the RAG-2(-/-) was remarkably preserved. Correspondingly, anti-donor proliferative and humoral immune responses were undetectable in C3H --> RAG-2(-/-) recipients as was the intragraft mRNA expression of the Th(1) and the Th(2)-type cytokines. CONCLUSIONS: Taken together, these data suggest that in this murine model of aortic allotransplantation, donor-specific cellular and humoral responses play a dominant role in the initiation and perpetuation of chronic rejection.

Aging increases p16 INK4a expression in vascular smooth-muscle cells.

Alteration of VSMC (vascular smooth-muscle cell) physiology is associated with the development of atherosclerosis and restenosis. We hypothesize that aging up-regulates the expression of p16 INK4a in VSMCs, which may increase the susceptibility of blood vessels to vascular occlusive diseases. Aortic VSMCs were obtained from young and aged mice. Cells from aged mice grew more slowly than those from their younger counterparts. Progression of cell cycle in response to serum stimulation was significantly inhibited in those cells with aging, as determined by FACS after propidium iodide staining. A significant up-regulation of p16 INK4a (2.5-fold, P=0.0012) was found in VSMC from aged animals using gene arrays. The up-regulation of this gene was further confirmed by quantitative RT-PCR (reverse transcription-PCR) and Western-blot experiments. Immunostaining for p16 INK4a confirmed that aortas from aged mice contained more p16 INK4a+ SMA (smooth-muscle cell actin)+ cells than aortas from young animals (26.79+/-2.45 versus 7.06+/-1.44, P=0.00027, n=4). In conclusion, we have shown that aging up-regulates the expression of p16 INK4a in VSMC in both cultures and arteries. The increase in p16 INK4a in the vasculature with aging may modify VSMC's response to post-injury stress and therefore accelerate the development of age-related cardiovascular diseases.

Nicotinic and PDGF-receptor function are essential for nicotine-stimulated mitogenesis in human vascular smooth muscle cells.

Cigarette smoking is implicated in the formation of occlusive vascular diseases. Nicotine's role in this process is incompletely understood. Nicotine's effect on human aortic vascular smooth muscle cells (HaVSMC) and the role of the nicotinic receptor (nAChR), platelet-derived growth factor (PDGF), and the PDGF-receptor (PDGF-R) in this response were studied. Nicotine's mitogenic effect was characterized by three methods: thymidine incorporation, a viability/proliferation assay based on metabolic conversion of tetrazolium salt to formazan dye and cell counting. Nicotine administration (10(-6) M) stimulated cell cycle entry marked by increased DNA synthesis, PCNA and cyclin D1 production, and increased cell division. Nicotinic receptor blockade with d-tubocurarine, a nicotinic AchR blocker, decreased nicotine-induced DNA synthesis, and cell division (0.33 +/- 0.04, 0.77 +/- 0.31-fold decrease, respectively). Nicotine increased cellular PDGF-BB transcript levels and protein release (ELISA: 1.6 +/- 0.5-fold increase) but not PDGF-AA or PDGF-AB release. Nicotine increased PDGFbeta-receptor protein content. PDGF inactivation with anti-PDGF antibody abolished nicotine-induced DNA synthesis (1.9 +/- 0.08-fold decrease). PDGF-R blockade with the PDGF-R antagonist tyrphostin AG 1295 decreased nicotine-induced DNA synthesis and cell division (0.25 +/- 0.01, 0.44 +/- 0.2-fold decrease, respectively). PDGF-R blockade reversed nicotine-stimulated increases in PDGF release, PDGF-BB transcripts, and PDGF-receptor levels (0.68 +/- 0.34; 0.46 +/- 0.01; 0.28 +/- 0.01-fold decrease, respectively). In conclusion, nicotine-mediated activation of nAChRs increases PDGF-BB transcription and protein production as well as PDGF beta-receptor levels. PDGF-BB/PDGF-R interaction is vital in nicotine's mitogenic actions on human aortic smooth muscle cells.

Molecular dissection of mouse soluble guanylyl cyclase alpha1 promoter.

Soluble guanylyl cyclase (sGC) is the only known receptor for nitric oxide (NO) and is downregulated in aging and hypertension. Little is known about sGC gene transcriptional regulation. In order to characterize the sGC transcriptional system, we cloned and sequenced the 5(') flanking region of mouse sGC alpha(1) gene (AY116663). Structurally, it is a non-canonical TATA-less promoter that we mapped to chromosome 3 with many putative regulation sites for Sp-1, NF-kappaB, and AP-1 transcription factors amongst others, and two (TG:CA)(n) dinucleotide microsatellites near the transcriptional start point. The cloned upstream sequence produced a 5-fold increase in luciferase activity in Cos7, HeLa, NIH3T3, and 293 cells as well as in mouse VSMC-like kidney mesangial cells. In the latter cell type, we showed that sGC alpha(1) promoter activity was dependent on the presence of its 5(') unstranslated region (5(')UTR).

Aging exacerbates neointimal formation, and increases proliferation and reduces susceptibility to apoptosis of vascular smooth muscle cells in mice.

OBJECTIVES: In response to injury, aging mediates exaggerated neointimal formation, the pathologic hallmark of obliterative vascular diseases. We assessed the development of neointima in a model of mechanical vascular injury in aging mice (18 months old) and young mice (2 months old). To investigate the mechanisms by which aging affects neointimal formation, we also carried out a set of in vitro studies to characterize the biologic properties of vascular smooth muscle cells (VSMCs) derived from aging and young mice. METHODS: Aging and young mice were subjected to wire injury to the carotid artery. Four weeks later injured arteries were harvested, and neointimal formation was histologically assessed. The profiles of angiogenesis-related genes between aortic VSMCs derived from aging and young mice were compared with complementary DNA arrays. Expression of platelet-derived growth factor receptor-alpha (PDGFR-alpha) and proliferation in response to platelet-derived growth factor-BB (PDGF-BB) by VSMCs were assessed. Susceptibility to apoptosis in aging and young VSMCs in response to nitric oxide and serum starvation was investigated. In addition, the level of apoptosis in neointimal VSMCs (by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay) was compared between aging and young animals. RESULTS: When compared with young mice, aging mice exhibited exaggerated neointimal formation (intima-media ratio, 1.17 +/- 0.57 vs 0.49 +/- 0.16; P < .0001). Aging VSMCs expressed higher levels of PDGFR-alpha (12.0% +/- 2.7% vs 3.2 +/- 0.67%; P = .034) and greater proliferative response (4-fold increase) to PDGF-BB, compared with young VSMCs. However, aging VSMCs were less susceptible to apoptosis when subjected to serum starvation (75% less) and exposure to nitric oxide (50% less). Furthermore, there was more apoptosis in the neointima of young arteries than in their aging counterparts (8.75% +/- 3.3% vs 2.8% +/- 1.9; P = .021). CONCLUSIONS: Age-dependent increases in PDGFR-alpha may alter VSMC proliferation, and when coupled with resistance to apoptosis could contribute to exaggerated neointima formation in aging animals. Of significance, our findings in the mouse will enable application of abundant molecular tools afforded by this species to further dissect the mechanisms of exaggerated neointimal formation associated with aging. CLINICAL RELEVANCE: Neointimal formation is the pathologic hallmark of obliterative vascular diseases, including primary atherosclerosis, post stent restenosis, graft occlusion after vascular bypass procedures, and transplant allograft vasculopathy. Aging is an independent risk factor for development of cardiovascular diseases, and aging exaggerates neointimal formation after vascular injury. Understanding the mechanisms responsible for this phenomenon may facilitate prevention or provide new therapies for vascular occlusive diseases, which are so prevalent in the aging population. Our ability to reproduce the model in the mouse will no doubt facilitate such understanding.

Effects of ventricular unloading on apoptosis and atrophy of cardiac myocytes.

BACKGROUND: Ventricular unloading decreases cardiac ventricular mass. This loss of ventricular mass can be due to either atrophy (a reversible process) or apoptosis (an irreversible process) of the cardiac myocytes. We investigated the effect of ventricular unloading on atrophy and apoptosis of cardiac myocytes, using working and nonworking transplant heart models in rats. MATERIALS AND METHODS: ACI rats underwent heterotopic heart transplantation with two different techniques to create working and nonworking cardiac grafts. Cardiac grafts were harvested at different time points after transplantation. TUNEL, caspase-3 assay, and electron microscopy were used to assess the degree of apoptosis while cellular atrophy was estimated by calculation of the cytoplasmic index (CI = mean sectional cytoplasmic area/nucleus). RESULTS: Ventricular mass reduction was more pronounced in nonworking than in working hearts (P < 0.05). Apoptotic index and caspase-3 activities increased in both groups, peaking at 3 days after transplantation, but were not significantly different between the two models. The cytoplasmic index was significantly lower in nonworking than in working grafts (P < 0.05). CONCLUSIONS: These data suggest that cellular atrophy is the primary mechanism that accounts for myocardial weight reduction following ventricular unloading. The inference is that ventricular unloading by ventricular assist devices may not cause permanent loss of cardiac myocytes, thus allowing for functional recovery.