SRT2183 and SRT1720, but not Resveratrol, Inhibit Osteoclast Formation and Resorption in the Presence or Absence of Sirt1.

Background: Osteoclastic bone resorption markedly increases with aging, leading to osteoporosis characterized by weak and fragile bones. Mice exhibit greater bone resorption and poor bone mass when Sirt1 is removed from their osteoclasts. Here we investigated the ex vivo impacts of putative Sirt1 activators, Resveratrol (RSV), SRT2183, and SRT1720, on osteoclast formation and activity in primary mouse bone marrow cells (BMCs) derived from wild-type (WT) and osteoclast specific Sirt1 knockout (OC-Sirt1KO) mice and in the RAW264.7 mouse macrophage cell line. Results: We found that SRT2183 and SRT1720 inhibit the formation of osteoclasts and actin belts in BMCs and RAW264.7 cells, whereas RSV does not. We also observed that the OC-Sirt1KO mice exhibited less bone mineral density, and the BMCs harvested from these mice yielded more osteoclasts than BMCs harvested from littermate controls. Interestingly, both SRT2183 and SRT1720 reduced osteoclast and actin belt formation in BMCs from OC-Sirt1KO mice. SRT2183 and SRT1720 also significantly disrupted actin belts of mature osteoclasts generated from BMCs of WT mice, within 3 and 6 hours of administration, respectively. Furthermore, these compounds inhibited the resorption activity of mature osteoclasts, while RSV did not. Conclusion: Our findings suggest SRT2183 and SRT1720 impede bone resorption by disrupting actin belts of mature osteoclasts, inhibit actin belt formation, and inhibit osteoclastogenesis even in the absence of Sirt1. Thus, the mechanism of action of these compounds appears to extend beyond Sirt1 activation and possibly pave the way for potential new therapies in alleviating osteoporosis associated bone loss.

Vitamin D Insufficiency Reduces Grip Strength, Grip Endurance and Increases Frailty in Aged C57Bl/6J Mice.

Low 25-OH serum vitamin D (VitD) is pervasive in older adults and linked to functional decline and progression of frailty. We have previously shown that chronic VitD insufficiency in "middle-aged" mice results in impaired anaerobic exercise capacity, decreased lean mass, and increased adiposity. Here, we examine if VitD insufficiency results in similar deficits and greater frailty progression in old-aged (24 to 28 months of age) mice. Similar to what we report in younger mice, older mice exhibit a rapid and sustained response in serum 25-OH VitD levels to differential supplementation, including insufficient (125 IU/kg chow), sufficient (1000 IU/kg chow), and hypersufficient (8000 IU/kg chow) groups. During the 4-month time course, mice were assessed for body composition (DEXA), physical performance, and frailty using a Fried physical phenotype-based assessment tool. The 125 IU mice exhibited worse grip strength (p = 0.002) and inverted grip hang time (p = 0.003) at endpoint and the 8000 IU mice transiently displayed greater rotarod performance after 3 months (p = 0.012), yet other aspects including treadmill performance and gait speed were unaffected. However, 125 and 1000 IU mice exhibited greater frailty compared to baseline (p = 0.001 and p = 0.038, respectively), whereas 8000 IU mice did not (p = 0.341). These data indicate targeting higher serum 25-OH vitamin D levels may attenuate frailty progression during aging.

AP-1 and Mitf interact with NFATc1 to stimulate cathepsin K promoter activity in osteoclast precursors.

Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption and osteoporotic bone loss. We have previously shown that activator protein 1 (AP-1) stimulates CTSK promoter activity and that proximal nuclear factor of activated T cells cytoplasmic 1 (NFATc1)-binding sites play a major role in the stimulation of CTSK gene expression by receptor activator of NFκB ligand (RANKL). In the present study, we have extended these observations and further dissected the effects of transcription factors involved in the regulation of CTSK gene expression. Our aim was to investigate the cooperative interplay among transcription factors AP-1, microphthalmia-associated transcription factor (Mitf), and NFATc1, and the consequent regulatory effects on CTSK transcription. Experiments were carried out in RAW 264.7 cells, which can be readily differentiated to osteoclasts upon RANKL stimulation. Our data show that AP-1, Mitf, and NFATc1 are capable of independently stimulating CTSK promoter activity. A combination of any two factors further enhances CTSK promoter activity, with the combination of AP-1 (c-fos/c-jun) and NFATc1 inducing the largest increase. We further identify a synergistic effect when all three factors cooperate intimately at the proximal promoter region, yielding maximal transcriptional upregulation of the CTSK promoter. RANKL induces temporal localization of AP-1 and NFATc1 to the CTSK promoter. These results suggest that the interaction of multiple transcription factors mediate a maximal response to RANKL-induced CTSK gene expression.

Treatment with an inhibitor of fatty acid synthase attenuates bone loss in ovariectomized mice.

Bone and fat cells have an antagonistic relationship. Adipocytes exert a toxic effect on bone cells in vitro through the secretion of fatty acids, which are synthesized by fatty acid synthase (FAS). Inhibition of FAS in vitro rescues osteoblasts from fat-induced toxicity and cell death. In this study, we hypothesized that FAS inhibition would mitigate the loss of bone mass in ovariectomized (OVX) mice. We treated OVX C57BL/6 mice with cerulenin (a known inhibitor of FAS) for 6 weeks and compared their bone phenotype with vehicle-treated controls. Cerulenin-treated mice exhibited a significant decrease in body weight, triglycerides, leptin, and marrow and subcutaneous fat without changes in serum glucose or calciotropic hormones. These effects were associated with attenuation of bone loss and normalization of the bone phenotype in the cerulenin-treated OVX group compared to the vehicle-treated OVX group. Our results demonstrate that inhibition of FAS enhances bone formation, induces uncoupling between osteoblasts and osteoclasts, and favors mineralization, thus providing evidence that inhibition of FAS could constitute a new anabolic therapy for osteoporosis.

Chronic vitamin D insufficiency impairs physical performance in C57BL/6J mice.

Vitamin D insufficiency (serum 25-OH vitamin D < 30 ng/ml) affects 70-80% of the general population, yet the long-term impacts on physical performance and the progression of sarcopenia are poorly understood. We therefore followed 6-month-old male C57BL/6J mice (n=6) consuming either sufficient (STD, 1000 IU) or insufficient (LOW, 125 IU) vitamin D3/kg chow for 12 months (equivalent to 20-30 human years). LOW supplemented mice exhibited a rapid decline of serum 25-OH vitamin D levels by two weeks that remained between 11-15 ng/mL for all time points thereafter. After 12 months LOW mice displayed worse grip endurance (34.6 ± 14.1 versus 147.5 ± 50.6 seconds, p=0.001), uphill sprint speed (16.0 ± 1.0 versus 21.8 ± 2.4 meters/min, p=0.0007), and stride length (4.4 ± 0.3 versus 5.1 ± 0.3, p=0.002). LOW mice also showed less lean body mass after 8 months (57.5% ± 5.1% versus 64.5% ± 4.0%, p=0.023), but not after 12 months of supplementation, as well as greater protein expression of atrophy pathway gene atrogin­1. Additionally, microRNA sequencing revealed differential expression of mIR­26a in muscle tissue of LOW mice. These data suggest chronic vitamin D insufficiency may be an important factor contributing to functional decline and sarcopenia.

High Intensity Interval Training Improves Physical Performance and Frailty in Aged Mice.

Sarcopenia and frailty are highly prevalent in older individuals, increasing the risk of disability and loss of independence. High intensity interval training (HIIT) may provide a robust intervention for both sarcopenia and frailty by achieving both strength and endurance benefits with lower time commitments than other exercise regimens. To better understand the impacts of HIIT during aging, we compared 24-month-old C57BL/6J sedentary mice with those that were administered 10-minute uphill treadmill HIIT sessions three times per week over 16 weeks. Baseline and end point assessments included body composition, physical performance, and frailty based on criteria from the Fried physical frailty scale. HIIT-trained mice demonstrated dramatic improvement in grip strength (HIIT 10.9% vs -3.9% in sedentary mice), treadmill endurance (32.6% vs -2.0%), and gait speed (107.0% vs 39.0%). Muscles from HIIT mice also exhibited greater mass, larger fiber size, and an increase in mitochondrial biomass. Furthermore, HIIT exercise led to a dramatic reduction in frailty scores in five of six mice that were frail or prefrail at baseline, with four ultimately becoming nonfrail. The uphill treadmill HIIT exercise sessions were well tolerated by aged mice and led to performance gains, improvement in underlying muscle physiology, and reduction in frailty.

A mouse model of vitamin D insufficiency: is there a relationship between 25(OH) vitamin D levels and obesity?

BACKGROUND: Vitamin D insufficiency (serum 25-OH vitamin D > 10 ng/ml and < 30 ng/ml) is prevalent in the obese (body mass index (BMI) > 30 kg/m2), yet relationships between the two are poorly understood. Objectives of this study include identification of the impact of obesity on reducing serum 25-OH vitamin D concentration, particularly in response to altered vitamin D3 supplementation, and to elucidate the longitudinal impact of serum 25-OH vitamin D on body mass index. METHODS: Twenty four-week-old lean and obese male C57BL/6 J mice were fed low, standard, or high levels of cholecalciferol supplementation and followed for 24 weeks. Longitudinal measurements include serum 25-OH and 1,25-(OH)2 vitamin D, intact PTH, and calcium concentrations, as well as BMI, bone density and body fat/lean mass. RESULTS: Baseline serum 25-OH concentrations were not different in lean and obese mice (lean 32.8 ± 4.4 ng/ml versus obese 30.9 ± 1.6 ng/ml p = 0.09). Lean mice receiving low supplementation exhibited rapid declines in serum 25-OH vitamin D concentrations, falling from 33.4 ± 5.4 ng/ml to 14.5 ± 3.4 ng/ml after 2 weeks, while obese mice declined at a lower rate, falling from 30.9 ± 1.5 to 19.0 ± 0.9 ng/ml within the same time period. Surprisingly, high vitamin D3 supplementation did not substantially increase serum vitamin D concentrations above standard supplementation, in either lean or obese mice. No differences in serum 1,25-(OH)2 vitamin D, intact parathyroid hormone (PTH) or serum calcium were observed between lean and obese mice within the same vitamin D supplementation group. Yet obese mice exhibited lower serum calcitriol, higher serum PTH, and lower bone mineral density (BMD) than did lean mice. Additionally, neither body mass index nor body fat % was significantly correlated with vitamin D concentrations. Interestingly, lean mice with high vitamin D supplementation consumed significantly more food than did lean mice with standard or low supplementation (14.6 ± 1.7 kcal/mouse/day versus 11.8 ± 1.4 and 12.3 ± 1.7 respectively, p < 0.0001 for both). CONCLUSIONS: Low cholecalciferol supplementation in both lean and obese mice significantly and sustainably reduces serum 25-OH vitamin D concentrations. Interestingly, obesity slowed the rate of decline. Over the period of the study, vitamin D insufficiency was not subsequently correlated with greater BMI/body fat, although lean mice with high supplementation consumed greater calories with no apparent BMI increase.

Hybrid Biomaterial with Conjugated Growth Factors and Mesenchymal Stem Cells for Ectopic Bone Formation.

Bone is a highly vascularized tissue and efficient bone regeneration requires neovascularization, especially for critical-sized bone defects. We developed a novel hybrid biomaterial comprising nanocalcium sulfate (nCS) and fibrin hydrogel to deliver mesenchymal stem cells (MSCs) and angiogenic factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 9 (FGF9), to promote neovascularization and bone formation. MSC and growth factor(s)-loaded scaffolds were implanted subcutaneously into mice to examine their angiogenic and osteogenic potential. Micro CT, alkaline phosphatase activity assay, and histological analysis were used to evaluate bone formation, while immunohistochemistry was employed to assess neovessel formation. The presence of fibrin preserved the nCS scaffold structure and promoted de novo bone formation. In addition, the presence of bone morphogenic protein 2-expressing MSC in nCS and fibrin hydrogels improved bone regeneration significantly. While FGF9 alone had no significant effect, the combination FGF9 and VEGF conjugated in fibrin enhanced neovascularization and bone formation more than 4-fold compared to nCS with MSC. Overall, our results suggested that the combination of nCS (to support bone formation) with a fibrin-based VEGF/FGF9 release system (support vascular formation) is an innovative and effective strategy that significantly enhanced ectopic bone formation in vivo.

Pro-angiogenic efficacy of transplanting endothelial progenitor cells for treating hindlimb ischemia in hyperglycemic rabbits.

AIMS: To evaluate the effectiveness of endothelial progenitor cells (EPCs) therapy in ischemia with or without hyperglycemia. METHODS: Japanese White Rabbits were randomly assigned to three groups, group SH, hyperglycemia with sham therapy (n=10); group NE, normoglycemia with autologous EPCs transplantation therapy (n=12); and group HE, hyperglycemia with autologous EPCs transplantation therapy (n=12). Hyperglycemia was induced by injecting alloxan and sustained for 12weeks. Hindlimb ischemia was induced by complete excision of the femoral artery. Ex vivo-expanded EPCs were derived from autologous bone marrow and transplanted intermuscularily in the ischemic hindlimb. Fourteen days after transplantation, the indicators were determined. RESULTS: There is no difference of the functions of ex vivo-expanded EPCs from autologous bone marrow between normoglycemic and hyperglycemic groups. We found significant improvement in both EPCs transplantation therapy groups compared to sham, in terms of the angiogenesis index (8.62±1.36, 11.12±2.23, 12.35±2.97), capillary density (7.06±0.91, 13.51±1.16, 13.90±2.78), capillary to muscle fiber ratio (0.68±0.09, 0.96±0.11,0.89±0.10), muscle VEGF expression (0.22±0.07, 0.41±0.08, 0.38±0.07ng/g). We found no significant differences between hyperglycemic and normoglycemic EPCs therapy groups except for 5 pro-angiogenic genes that were upregulated in HE as compared to NE. CONCLUSION: Ex vivo expanded EPCs from autologous bone marrow transplantation is an effective therapeutic method for hindlimb ischemia in rabbits regardless of glycemic state.

Retinoic acid inhibits NFATc1 expression and osteoclast differentiation.

Ingestion of excess vitamin A appears to correlate with an increased fracture risk, an outcome that is likely mediated by retinoic acids (RAs); these are vitamin A metabolites that have dramatic effects on skeletal development. We studied the impacts of RA and isoform-specific RA receptor (RAR) agonists (α, ß, and γ) on osteoclast formation (osteoclastogenesis) in two model systems: RAW264.7 cells and murine bone marrow-derived monocytes. The pan-RAR agonists, all-trans and 9-cis RA, inhibited receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclast differentiation in a concentration-dependent manner. Isoform-specific RAR agonists (α, ß, and γ) also inhibited osteoclastogenesis, with the RARα agonist producing the most consistent reductions in both osteoclast number and size and total area covered. Inhibition of osteoclastogenesis correlated with reductions in expression, DNA binding, and nuclear abundance of nuclear factor of activated T cells c1 (NFATc1), a transcription factor critical for osteoclastogenesis. The upregulation of three NFATc1-responsive genes, cathepsin K, dendritic cell-specific transmembrane protein and osteoclast-associated receptor were similarly reduced following RA or RAR agonist exposure. These results suggest that RA blocks in vitro RANKL-mediated osteoclastogenesis by decreasing NFATc1 function.

Identification of NFAT binding sites that mediate stimulation of cathepsin K promoter activity by RANK ligand.

The receptor activator of NFkappaB ligand (RANKL) is a critical mediator of osteoclastogenesis and regulates cathepsin K (CTSK) expression, which is essential for normal bone resorption. RANKL acts, in part, via the Ca(2+)/calmodulin/calcineurin signaling pathway, which in turn, activates NFATc1 (nuclear factor of activated T-cells) and downstream gene expression. We investigated the signals and promoter elements that regulate CTSK gene expression in RAW 264.7 cells, which can be differentiated to osteoclasts by RANKL. Disrupting Ca(2+) signaling, by blocking Ca(2+) channels, thus inhibiting calcineurin or chelation of intracellular Ca(2+), prevented the stimulation of CTSK expression by RANKL. Both RANKL treatment and overexpression of NFATc1 dramatically enhanced CTSK promoter activity, but not in an identical manner. NFATc1 regulates CTSK promoter activity, but the motifs have not been explicitly identified. We found that as few as 238 bp of the CTSK promoter were sufficient to elicit a marked response to both RANKL and NFATc1, truncations of the CTSK promoter illustrated differences in regional responsiveness. Transfection analysis of CTSK promoter-luciferase plasmids revealed that NFATc1 binding sites at 85, 289 and 345 bp upstream of the transcriptional start site mediated responses to RANKL and NFATc1. Deletion of a 4-bp core element from the site at -85 bp dramatically reduced the response of the CTSK promoter to both RANKL and NFATc1, whereas a similar deletion at -345 bp decreased NFATc1- but not RANKL-mediated responses. Mutation of the site at -289 bp did not affect NFAT-mediated stimulation of CTSK on its own, but did decrease responsiveness in combination with either or both of the other two deletions. Electrophoretic mobility shift assays demonstrated NFATc1 binding to oligonucleotides containing the -85-bp and -345-bp sites, while chromatin immunoprecipitation assays demonstrated enhanced in situ binding by NFATc1 to two analogous sites in the mouse CTSK promoter in response to RANKL treatment. Therefore, proximal NFAT binding sites play a significant role in the NFATc1-mediated stimulation of CTSK gene expression by RANKL.

AP-1 stimulates the cathepsin K promoter in RAW 264.7 cells.

Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption, and CTSK levels increase with osteoclast differentiation and activation, a process that is controlled by a complex physiological network of hormones and cytokines. A critical regulator of this process is receptor activator of NF-kappaB ligand (RANKL), a member of the tumor necrosis factor (TNF) superfamily of cytokines that can act via the TNF receptor activating factor (TRAF6)/AP-1 signaling pathway. However, the mechanism whereby RANKL modulates CTSK expression is not fully understood. Therefore, we investigated the regulation of CTSK expression and promoter activity in RAW 264.7 osteoclast precursor cells, which can be readily differentiated to osteoclasts upon RANKL stimulation. Western blot analysis, real-time RT-PCR and luciferase reporter gene assays revealed that RANKL stimulated CTSK expression and promoter activity in a dose- and time-dependent manner and that this activation was inhibited by either dominant negative (DN) TRAF6 or DN-c-fos. TRAF6 stimulated the basal activity of a truncated CTSK promoter, and DN-c-fos blocked this stimulation. JunB alone also stimulated basal CTSK promoter activity, whereas c-jun, JunD or c-fos alone did not. However, co-transfection of any of these jun-family members with c-fos (AP-1) significantly increased CTSK promoter expression. siRNA targeted against c-jun or junB suppressed RANKL-mediated CTSK expression. Therefore, both TRAF6 and AP-1 help regulate the basal and RANKL-mediated stimulation of CTSK gene expression in RAW 264.7 cells.

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.

RANK ligand and interferon gamma differentially regulate cathepsin gene expression in pre-osteoclastic cells.

Receptor activator of NF-kappaB ligand (RANKL) and interferon gamma (IFN-gamma) are critical and opposing mediators of osteoclastogenesis, exerting stimulatory and inhibitory effects, respectively. Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption. We have examined the role of IFN-gamma in the regulation of CTSK expression in the murine monocytic RAW 264.7 cell line, which can be readily differentiated to bone-resorbing osteoclasts upon RANKL treatment. Real-time RT-PCR reveals that RANKL stimulates CTSK mRNA expression in a dose- and time-dependent fashion, but that RANKL does not alter the expression of cathepsin L (CTSL) and cathepsin S (CTSS) mRNA. IFN-gamma stimulates both CTSL and CTSS expression after 3 days, but fails to significantly alter CTSK expression. IFN-gamma markedly inhibits the stimulation of CTSK mRNA and protein by RANKL, whereas RANKL suppresses the stimulation of CTSL and CTSS mRNA by IFN-gamma. IFN-gamma also ablates the RANKL induced osteoclastic differentiation of RAW cells. In RAW cells stably transfected with a CTSK promoter-luciferase plasmid containing the 1618 bp upstream of the transcription initiation site, IFN-gamma inhibits CTSK promoter activity and ablates its induction by RANKL. In conclusion, IFN-gamma and RANKL differentially regulate cathepsin K, S, and L gene expression in pre-osteoclastic cells, and there appears to be significant cross talk between the signal transduction pathways mediating the responses to RANKL and IFN-gamma.

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.

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).