Decorin antagonizes IGF receptor I (IGF-IR) function by interfering with IGF-IR activity and attenuating downstream signaling

We have recently discovered that the insulin-like growth factor receptor I (IGF-IR) is up-regulated in human invasive bladder cancer and promotes migration and invasion of transformed urothelial cells. The proteoglycan decorin, a key component of the tumor stroma, can positively regulate the IGF-IR system in normal cells. However, there are no available data on the role of decorin in modulating IGF-IR activity in transformed cells or in tumor models. Here we show that the expression of decorin inversely correlated with IGF-IR expression in low and high grade bladder cancers (n = 20 each). Decorin bound with high affinity IGF-IR and IGF-I at distinct sites and negatively regulated IGF-IR activity in urothelial cancer cells. Nanomolar concentrations of decorin promoted down-regulation of IRS-1, one of the critical proteins of the IGF-IR pathway, and attenuated IGF-I-dependent activation of Akt and MAPK. This led to decorin-evoked inhibition of migration and invasion upon IGF-I stimulation. Notably, decorin did not cause down-regulation of the IGF-IR in bladder, breast, and squamous carcinoma cells. This indicates that decorin action on the IGF-IR differs from its known activity on other receptor tyrosine kinases such as the EGF receptor and Met. Our results provide a novel mechanism for decorin in negatively modulating both IGF-I and its receptor. Thus, decorin loss may contribute to increased IGF-IR activity in the progression of bladder cancer and perhaps other forms of cancer where IGF-IR plays a role.     

A role for T lymphocytes in mediating cardiac diastolic function

The induction of T helper (TH) lymphocytes by distinct TH ligands results in a differentiation to TH1/TH2 subsets based on their unique pattern of cytokine secretion and effector functions. We hypothesized that the relative proportion of TH1/TH2 directly relates to cardiac fibroblast (CF) function and thereby cardiac extracellular matrix (ECM) composition and cardiac diastolic function in the absence of injury or altered wall stress. We compared the effect of selective TH1 with TH2 inducers on cardiac gene expression, ECM composition, and diastolic function in C57BL/J mice. Twelve weeks after immune modulation, the left ventricular stiffness (beta) was significantly increased in the TH1 group and decreased in the TH2 group (P < 0.01). The TH2 group also demonstrated significantly increased end-diastolic and end-systolic volumes (P < 0.01). Cardiac gene expression patterns for pro-matrix metalloproteinase (MMP)-9 and -13 were increased by greater than fivefold in the TH2 group and significantly decreased in the TH1 group (P < 0.05). The total cardiac collagen and cross-linked collagen were significantly increased in the TH1 group and decreased in the TH2 group (P < 0.01). Coculturing lymphocytes harvested from the treated mice with naive primary CF demonstrated a direct control of the lymphocytes on CF pro-collagen, pro-MMP gene expression, and MMP activity. These results suggest that the TH phenotype differentially affects diastolic function through modulating CF pro-collagen and pro-MMP gene expression, MMP activity, and cardiac collagen cross-linking, resulting in altered ECM composition. Thus modulation of TH lymphocyte function could promote adaptive remodeling in heart failure and postmyocardial infarction.     

Peroxynitrite irreversibly decreases diastolic and systolic function in cardiac muscle

Much of the damaging action of nitric oxide in heart may be due to its diffusion-limited reaction with superoxide to form peroxynitrite. Direct infusion of peroxynitrite into isolated perfused hearts fails to model the effects of in situ formation because the bulk of peroxynitrite decomposes before reaching the myocytes. To examine the direct effects of peroxynitrite on the contractile apparatus of the heart, we exposed intact and skinned rat papillary muscles to a steady state concentration of 4-microM peroxynitrite for 5 min, followed by a 30-min recovery period to monitor irreversible effects. In intact muscles developed force fell immediately to 26% of initial force, recovering to 43% by 30 min. Resting tension increased by 600% immediately, and was still elevated 500% by 30 min. Nitrotyrosine immunochemistry showed that peroxynitrite can induce tyrosine nitration at low concentrations and is capable of penetrating 200-380 microm into the papillary muscle after a 5-min infusion. Decomposed peroxynitrite had no effect on either intact or skinned muscle developed force or resting tension. Our results show that peroxynitrite directly damages both developed force and resting tension of isolated heart muscle, which can be extrapolated to systolic and diastolic injury in intact hearts.     

Overexpression of glutathione peroxidase attenuates myocardial remodeling and preserves diastolic function in diabetic heart

Oxidative stress plays an important role in the structural and functional abnormalities of diabetic heart. Glutathione peroxidase (GSHPx) is a critical antioxidant enzyme that removes H(2)O(2) in both the cytosol and mitochondia. We hypothesized that the overexpression of GSHPx gene could attenuate left ventricular (LV) remodeling in diabetes mellitus (DM). We induced DM by injection of streptozotocin (160 mg/kg ip) in male GSHPx transgenic mice (TG+DM) and nontransgenic wildtype littermates (WT+DM). GSHPx activity was higher in the hearts of TG mice compared with WT mice, with no significant changes in other antioxidant enzymes. LV thiobarbituric acid-reactive substances measured in TG+DM at 8 wk were significantly lower than those in WT+DM (58 +/- 3 vs. 71 +/- 5 nmol/g, P < 0.05). Heart rate and aortic blood pressure were comparable between groups. Systolic function was preserved normal in WT+DM and TG+DM mice. In contrast, diastolic function was impaired in WT+DM and was improved in TG+DM as assessed by the deceleration time of peak velocity of transmitral diastolic flow and the time needed for relaxation of 50% maximal LV pressure to baseline value (tau; 13.5 +/- 1.2 vs. 8.9 +/- 0.7 ms, P < 0.01). The TG+DM values were comparable with those of WT+Control (tau; 7.8 +/- 0.2 ms). Improvement of LV diastolic function was accompanied by the attenuation of myocyte hypertrophy, interstitial fibrosis, and apoptosis. Overexpression of GSHPx gene ameliorated LV remodeling and diastolic dysfunction in DM. Therapies designed to interfere with oxidative stress might be beneficial to prevent cardiac abnormalities in DM.     

Proteolytic N-terminal truncation of cardiac troponin I enhances ventricular diastolic function

Besides the core structure conserved in all troponin I isoforms, cardiac troponin I (cTnI) has an N-terminal extension that contains phosphorylation sites for protein kinase A under beta-adrenergic regulation. A restricted cleavage of this N-terminal regulatory domain occurs in normal cardiac muscle and is up-regulated during hemodynamic adaptation (Z.-B. Yu, L.-F. Zhang, and J.-P. Jin (2001) J. Biol. Chem. 276, 15753-15760). In the present study, we developed transgenic mice overexpressing the N-terminal truncated cTnI (cTnI-ND) in the heart to examine its biochemical and physiological significance. Ca(2+)-activated actomyosin ATPase activity showed that cTnI-ND myofibrils had lower affinity for Ca(2+) than controls, similar to the effect of isoproterenol treatment. In vivo and isolated working heart experiments revealed that cTnI-ND hearts had a significantly faster rate of relaxation and lower left ventricular end diastolic pressure compared with controls. The higher baseline relaxation rate of cTnI-ND hearts was at a level similar to that of wild type mouse hearts under beta-adrenergic stimulation. The decrease in cardiac output due to lowered preload was significantly smaller for cTnI-ND hearts compared with controls. These findings indicate that removal of the N-terminal extension of cTnI via restricted proteolysis enhances cardiac function by increasing the rate of myocardial relaxation and lowering left ventricular end diastolic pressure to facilitate ventricular filling, thus resulting in better utilization of the Frank-Starling mechanism.     

Cardiac-specific overexpression of insulin-like growth factor 1 attenuates aging-associated cardiac diastolic contractile dysfunction and protein damage

Aging is associated with hepatic growth hormone resistance resulting in a fall in serum insulin-like growth factor 1 (IGF-1) level. However, whether loss of IGF-1 contributes to cardiac aging is unclear. This study was designed to examine the effect of cardiac overexpression of IGF-1 on cardiomyocyte contractile function in young (3 mo) and old (26-28 mo) mice. Cardiomyocyte contractile function was evaluated, including peak shortening (PS), time to 90% PS, time to 90% relengthening (TR(90)), and maximal velocity of shortening/relengthening (+/-dL/dt). Levels of advanced glycation end product, protein carbonyl, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), phospholamban, and Na(+)/Ca(2+) exchanger were assessed by Western blot analysis. SERCA activity was measured by (45)Ca(2+) uptake. Aging induced a decline in plasma IGF-1 levels. Aged cells exhibited depressed +/-dL/dt, prolonged TR(90), and a steeper PS decline in response to increasing stimulus frequency compared with those in young myocytes. IGF-1 transgene alleviated aging-induced loss in plasma IGF-1 and aging-induced mechanical defects with little effect in young mice. The beneficial effect of IGF-1 transgene on aging-associated cardiomyocyte contractile dysfunction was somewhat mimicked by short-term in vitro treatment of recombinant IGF-1 (500 nM). Advanced glycation end product and protein carbonyl levels were higher in aged mice, which were not affected by IGF-1. Expression of SERCA2a (but not Na(+)/Ca(2+) exchanger and phospholamban) and SERCA activity were reduced with aging, which was ablated by the IGF-1 transgene. Collectively, our data suggest a beneficial role of IGF-1 in aging-induced cardiac contractile dysfunction, possibly related to improved Ca(2+) uptake.     

Dysregulation of protein kinase a signaling in the aged prefrontal cortex: new strategy for treating age-related cognitive decline

Activation of the cAMP/protein kinase A (PKA) pathway has been proposed as a mechanism for improving age-related cognitive deficits based on studies of hippocampal function. However, normal aging also afflicts prefrontal cortical cognitive functioning. Here, we report that agents that increase PKA activity impair rather than improve prefrontal cortical function in aged rats and monkeys with prefrontal cortical deficits. Conversely, PKA inhibition ameliorates prefrontal cortical cognitive deficits. Western blot and immunohistochemical analyses of rat brain further indicate that the cAMP/PKA pathway becomes disinhibited in the prefrontal cortex with advancing age. These data demonstrate that PKA inhibition, rather than activation, is the appropriate strategy for restoring prefrontal cortical cognitive abilities in the elderly.     

Silibinin attenuates cardiac hypertrophy and fibrosis through blocking EGFR‐dependent signaling

Cardiac hypertrophy is a major determinant of heart failure. The epidermal growth factor receptor (EGFR) plays an important role in cardiac hypertrophy. Since silibinin suppresses EGFR in vitro and in vivo, we hypothesized that silibinin would attenuate cardiac hypertrophy through disrupting EGFR signaling. In this study, we examined this hypothesis using neonatal cardiac myocytes and fibroblasts induced by angiotensin II (Ang II) and animal model by aortic banding (AB) mice. Our data revealed that silibinin obviously blocked cardiac hypertrophic responses induced by pressure overload. Meanwhile, silibinin markedly reduced the increased generation of EGFR. Moreover, these beneficial effects were associated with attenuation of the EGFR‐dependent ERK1/2, PI3K/Akt signaling cascade. We further demonstrated silibinin decreased inflammation and fibrosis by blocking the activation of NF‐κB and TGF‐β1/Smad signaling pathways in vitro and in vivo. Our results indicate that silibinin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through blocking EGFR activity and EGFR‐dependent different intracellular signaling pathways. J. Cell. Biochem. 110: 1111–1122, 2010. Published 2010 Wiley‐Liss, Inc.     

Pentyl-4-yn-valproic acid enhances both spatial and avoidance learning, and attenuates age-related NCAM-mediated neuroplastic decline within the rat medial temporal lobe

2-N-Pentyl-4-pentynoic acid [pentyl-4-yn-valproic acid (VPA)] is an analogue of valproic acid that induces neuritogenesis and increases neural cell adhesion molecule (NCAM) prevalence in cultured neural cells. As memory consolidation involves synapse growth, aided by cell adhesion molecule function, we determined whether or not pentyl-4-yn-VPA had cognition-enhancing properties. Pentyl-4-yn-VPA (16-85 mg/kg) significantly improved water maze learning and task retention when given prior to each training session. Acute administration of pentyl-4-yn-VPA also influenced memory consolidation processes as, when given at 3 h post-passive avoidance training, the amnesia induced by scopolamine given 6 h post-training was prevented in a dose-dependent manner. Chronic administration of pentyl-4-yn-VPA (16.8 or 50.4 mg/kg) also significantly reduced escape latencies in the water maze task, 24 h following the last drug administration. This improved spatial learning was accompanied by enhanced neuroplasticity as the expression of NCAM polysialylated neurons in the infragranular zone of the dentate gyrus and in layer II of the perirhinal and piriform cortex was increased significantly following chronic drug treatment. The cognition-enhancing qualities of pentyl-4-yn-VPA, combined with its ability to attenuate the age-related loss of the NCAM polysialylation state, suggest that it may effectively slow the onset of cognitive decline.     

Peak exercise stroke volume: associations with cardiac structure and diastolic function.

One of the most debilitating effects of primary aging is the decline in aerobic exercise capacity. One of its causes is an age-related decline in peak exercise stroke volume. This study's main purpose was to determine the cardiovascular adaptations to aging that most influence peak exercise stroke volume in the elderly. We hypothesized that increased left ventricular (LV) filling and mild concentric LV remodeling would be associated with an increase in peak exercise stroke volume corrected for lean body mass (LBM) and that an increased augmentation index (AI), which is a marker of arterial stiffness, would be associated with a decrease. A second aim was to determine the adaptations to aging that most influence LV concentric remodeling in the elderly. We hypothesized that AI would be a predictor of LV mass/LBM and the LV posterior wall thickness-to-LV radius ratio (h/r). We performed a cross-sectional study of cardiac and vascular adaptations to aging in 52 sedentary, elderly subjects. LV filling [as measured by the early-to-late transmitral flow velocity ratio (E/A)] was inversely correlated with and was an independent predictor of peak exercise stroke volume/LBM and was also a predictor of LV remodeling. AI was a predictor of LV remodeling (LV mass/LBM) but not of peak exercise stroke volume/LBM. We conclude that 1) maintenance of LV filling (E/A <1) is associated with a higher peak exercise stroke volume/LBM in very elderly subjects and thus may be a useful adaptation that enhances stroke volume during peak exercise, 2) LV remodeling and AI are less influential on peak exercise stroke volume/LBM, and 3) AI was the most important predictor of LV remodeling.     

Neurogenesis in a rat model of age-related cognitive decline

Age-related decrements in hippocampal neurogenesis have been suggested as a basis for learning impairment during aging. In the current study, a rodent model of age-related cognitive decline was used to evaluate neurogenesis in relation to hippocampal function. New hippocampal cell survival was assessed approximately 1 month after a series of intraperitoneal injections of 5-bromo-2'-deoxyuridine (BrdU). Correlational analyses between individual measures of BrdU-positive cells and performance on the Morris water maze task provided no indication that this measure of neurogenesis was more preserved in aged rats with intact cognitive abilities. On the contrary, among aged rats, higher numbers of BrdU-positive cells in the granule cell layer were associated with a greater degree of impairment on the learning task. Double-labelling studies confirmed that the majority of the BrdU+ cells were of the neuronal phenotype; the proportion of differentiated neurons was not different across a broad range of cognitive abilities. These data demonstrate that aged rats that maintain cognitive function do so despite pronounced reductions in hippocampal neurogenesis. In addition, these findings suggest the interesting possibility that impaired hippocampal function is associated with greater survival of newly generated hippocampal neurons at advanced ages.     

An analysis of the age-related decline in testicular steroidogenesis in the rat

The effect of aging in rats on serum and intratesticular testosterone levels, microsomal steroidogenic enzyme activities and microsomal cytochrome P-450 was studied. Serum testosterone levels were highest in 11-wk-old rats, declined at age 16 wk and further declined between ages 7 and 21 mo. Intratesticular testosterone levels in 21-mo-old rats were significantly lower than those of the other groups. The activity of 17 alpha-hydroxylase and C17-20 lyase, as well as cytochrome P-450, decreased significantly in 21-mo-old rats. The activity of 17 beta-hydroxysteroid oxidoreductase increased from 11 wk to 16 wk of age and then declined by 21 mo of age to the levels of 11-wk-old animals. Similar changes in delta 5-3,3-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase activities were observed, but were not statistically significant. These results suggest that the decline in testosterone production in old rats is predominantly a result of decreased oxygenase activity. Inasmuch as oxygenases are gonadotropin dependent, our results support the hypothesis that gonadotropin deficiency is the major factor responsible for Leydig cell dysfunction in old rats. Further, the decline in the ratio of 17 alpha-hydroxylase to C17-20 lyase with aging suggests that other factors affect these enzymes as well as the reduction in cytochrome P-450.     

Targeted deletion of Puma attenuates cardiomyocyte death and improves cardiac function during ischemia-reperfusion

The p53-upregulated modulator of apoptosis (Puma), a BH3-only member of the Bcl-2 protein family, is required for p53-dependent and -independent forms of apoptosis and has been implicated in the pathomechanism of several diseases, including cancer, acquired immunodeficiency syndrome, and ischemic brain disease. The role of Puma in cardiomyocyte death, however, has not been analyzed. On the basis of the ability of Puma to integrate diverse cell death stimuli, we hypothesized that Puma might be critical for cardiomyocyte death upon ischemia-reperfusion (I/R) of the heart. Here we show that hypoxia-reoxygenation of isolated cardiomyocytes led to an increase in Puma mRNA and protein levels. Moreover, if Puma was delivered by an adenoviral construct, cardiomyocytes died by apoptosis. Under ATP-depleted conditions, however, Puma overexpression primarily induced necrosis, suggesting that Puma is involved in the development of both types of cell death. Consistent with these findings, targeted deletion of Puma in a mouse model attenuated both apoptosis and necrosis. When the Langendorff ex vivo I/R model was used, infarcts were approximately 50% smaller in Puma(-/-) than in wild-type mice. As a result, after I/R, cardiac function was significantly better preserved in Puma(-/-) mice than in their wild-type littermates. Our study thus establishes Puma as an essential mediator of cardiomyocyte death upon I/R injury and offers a novel therapeutic target to limit cell loss in ischemic heart disease.     

Colchicine attenuates left ventricular hypertrophy but preserves cardiac function of aortic-constricted rats.

To investigate the effects of colchicine on left ventricular (LV) function and hypertrophy (LVH) of rats subjected to constriction of transverse aorta (TAoC), we evaluated SO (sham operated, vehicle; n = 25), SO-T (sham operated, colchicine 0.4 mg/kg body wt ip daily; n = 38), TAoC (vehicle; n = 37), and TAoC-T (TAoC, colchicine; n = 34) on the 2nd, 6th, and 15th day after surgery. Colchicine attenuated LVH of TAoC-T compared with TAoC rats, as evaluated by ratio between LV mass (LV(M)) and right ventricular mass, LV wall thickness, and average diameter of cardiac myocytes. Systolic gradient across TAoC ( approximately 45 mmHg), LV systolic pressure, LV end-diastolic pressure, and rate of LV pressure increase (+dP/dt) were comparable in TAoC-T and TAoC rats. However, the baseline and increases of LV systolic pressure-to-LV(M) and +dP/dt-to-LV(M) ratios induced by phenylephrine infusion were greater in TAoC-T and SO-T compared with SO rats. Baseline and increases of +dP/dt-to-LV(M) ratio were reduced in TAoC compared with SO rats. TAoC rats increased polymerized fraction of tubulin compared with SO, SO-T, and TAoC-T rats. Our results indicate that colchicine treatment reduced LVH to pressure overload but preserved LV function.