Gerald N. Audet

Temporal response of positive and negative regulators in response to acute and chronic exercise training in mice

•  Angiogenic regulators respond to acute exercise with different temporal expression patterns (e.g. 2–4 h versus 12–24 h) creating a complex multifaceted response that must be considered in studies using a single time point for post‐exercise analyses. •  In response to chronic training there appears to be a complex coordination in the proteomic responses of both positive and negative angiogenic factors that correspond with training‐induced muscle capillary adaptation, such that altered basal expression and acute responses to exercise appear to withdraw or reduce the stimulus of angiogenic regulators in an expanding capillary bed with active angiogenesis. •  These are the first data to show that nucleolin (a protein responsible for transcriptional processing and transportation of proteins from the cytoplasm to the nucleus) is responsive to acute exercise. We speculate that nucleolin may work in concert with vascular endothelial growth factor‐A (VEGF) and endostatin. •  Temporal responses observed in mice, particularly for VEGF, MMP‐2 and MMP‐9, may not be directly comparable to humans.

Expression of angiogenic regulators and skeletal muscle capillarity in selectively bred high aerobic capacity mice

Selective breeding for high voluntary wheel running in untrained mice has resulted in a ‘mini muscle’ (MM) phenotype, which has increased skeletal muscle capillarity compared with muscles from non‐selected control lines. Vascular endothelial growth factor (VEGF) and thrombospondin‐1 (TSP‐1) are essential mediators of skeletal muscle angiogenesis; thus, we hypothesized that untrained MM mice with elevated muscle capillarity would have higher basal VEGF expression and lower basal TSP‐1 expression, and potentially an exaggerated VEGF response to acute exercise. We examined skeletal muscle morphology and skeletal muscle protein expression of VEGF and TSP‐1 in male mice from two (untrained) mouse lines selectively bred for high exercise capacity (MM and Non‐MM), as well as one non‐selected control mouse line (normal aerobic capacity). In the MM mice, gastrocnemius (GA) and plantaris (PLT) muscle capillarity (i.e. capillary‐to‐fibre ratio and capillary density) were greater compared with control mice (P < 0.05). In Non‐MM mice, only muscle capillarity in PLT was greater than in control mice (P < 0.001). The soleus (SOL) showed no statistical differences in muscle capillarity among groups. In the GA, MM mice had 58% greater basal VEGF (P < 0.05), with no statistical difference in basal TSP‐1 when compared with control mice. In the PLT, MM mice had a 79% increase in basal VEGF (P < 0.05) and a 39% lower basal TSP‐1 (P < 0.05) compared with the control animals. Non‐MM mice showed no difference in basal VEGF in either the GA or the PLT compared with control mice. In contrast, basal TSP‐1 was elevated in the PLT, but not in the GA, of Non‐MM mice compared with control mice. Neither VEGF nor TSP‐1 was significantly different in SOL muscle among the three mouse lines. In response to acute exercise, MM mice displayed a 41 and 28% increase (P < 0.05) in VEGF in the GA and PLT, respectively, whereas neither control nor Non‐MM mice showed a significant VEGF response to acute exercise. In contrast, TSP‐1 levels were decreased by 90% in GA (P < 0.05) but increased by 50% in PLT (P < 0.05) in response to acute exercise in MM mice. The SOL showed no response to exercise for either VEGF or TSP‐1 for any of the mouse lines. These data, with the exception of the Non‐MM plantaris muscle, suggest that elevated capillarity is associated with altered balance between positive and negative angiogenic regulators (i.e. VEGF versus TSP‐1, respectively). Based on the greater capillarity and significant VEGF response to exercise in MM mice, these data suggest that VEGF expression may, at least in part, be genetically determined.

Chronic Delivery of a Thrombospondin-1 Mimetic Decreases Skeletal Muscle Capillarity in Mice

Angiogenesis is an essential process for normal skeletal muscle function. There is a growing body of evidence suggesting that thrombospondin-1 (TSP-1), a potent antiangiogenic protein in tumorigenesis, is an important regulator of both physiological and pathological skeletal muscle angiogenesis. We tested the hypothesis that chronic exposure to a TSP-1 mimetic (ABT-510), which targets the CD36 TSP-1 receptor, would decrease skeletal muscle capillarity as well as alter the balance between positive and negative angiogenic proteins under basal conditions. Osmotic minipumps with either ABT-510 or vehicle (5% dextrose) were implanted subcutaneously in the subscapular region of C57/BL6 mice for 14 days. When compared to the vehicle treated mice, the ABT-510 group had a 20% decrease in capillarity in the superficial region of the gastrocnemius (GA), 11% decrease in the plantaris (PLT), and a 35% decrease in the soleus (SOL). ABT-510 also decreased muscle protein expression of vascular endothelial growth factor (VEGF) in both the GA (−140%) and SOL (−62%); however there was no change in VEGF in the PLT. Serum VEGF was not altered in ABT-510 treated animals. Endogenous TSP-1 protein expression in all muscles remained unaltered. Tunnel staining revealed no difference in muscle apoptosis between ABT-510 and vehicle treated groups. These data provide evidence that the anti-angiogenic effects of TSP-1 are mediated, at least in part, via the CD36 receptor. It also suggests that under physiologic conditions the TSP-1/CD36 axis plays a role in regulating basal skeletal muscle microvessel density.

Altered hypothalamic inflammatory gene expression correlates with heat stroke severity in a conscious rodent model.

It has been suggested that heat-induced hypothalamic damage mediates core temperature (Tc) disturbances during heat stroke (HS) recovery; this is significant as hypothermia and/or fever have been linked to severity and overall pathological insult. However, to date there has been a lack of histological evidence in support of these claims. We hypothesized that local hypothalamic cytokines and/or chemokines, known regulators of Tc, are mediating the elevation in Tc during HS recovery even in the absence of histological damage. In experiment 1, the hypothalamus of Fischer 344 rats was examined for 84 cytokine/chemokine genes (real-time PCR) at multiple time points (Tc,Max, 1, 3, and 10 days) during mild HS recovery. In experiment 2, the hypothalamus of three different HS severities (MILD, moderate [MOD], and severe [SEV]) in rats were examined for the same genes as experiment 1 as well as six oxidative damage markers, at a single intermediate time point (1 day). Systemic cytokines were also analyzed in experiment 2 across the three severities. There were significant alterations in 25 cytokines/chemokines expression at Tc,Max, but little or no changes in expression at longer time points in experiment 1. In experiment 2 there were significant changes in gene expression in SEV rats only, with MILD and MOD rats showing baseline expression at 1 day, despite an absence of systemic cytokine expression in any severity. There was also no change in any oxidative marker of damage at 1 day, regardless of severity. In conclusion, we show only limited changes during long term recovery from HS, but demonstrate differences in hypothalamic gene expression patterns that may be driving HS pathology and morbidity. These findings contribute to our overall understanding of HS pathology in the CNS, as well as providing avenues for future pharmacological intervention.

Effects of detraining on the temporal expression of positive and negative angioregulatory proteins in skeletal muscle of mice

Skeletal muscle capillary regression is associated with elevated thrombospondin‐1 and vascular endothelial growth factor protein expression with detraining. Vascular endothelial growth factor and nucleolin responses to acute exercise are blunted in the triceps surae muscles following exercise training. In the plantaris and soleus muscles, this blunted response persists up to 28 days after cessation of training. Effects of detraining on skeletal muscle microvascular density appears to be similar among skeletal muscle of varying oxidative potential and is poorly associated with expression of matrix metalloproteinases‐2 and ‐9 and endostatin.

Abstract 3269: Basal VEGF protein expression in skeletal muscle, adipose tissue and mammary tumors of PyMT mice

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Obesity is associated with the overall risk of certain cancers, including breast cancer. The prevailing view is that metabolic alterations associated with obesity result in the production of excess vascular endothelial growth factor (VEGF) thereby establishing a local and systemic environment that favors the development and progression of breast cancer. We need to understand how VEGF from adipose is integrated with mammary tumor growth and vascular activity to modulate tumor progression. The MMTV-PyMT mouse is a clinically-relevant model for studying breast cancer progression as mammary tumors grow spontaneously over time; however, little is known about the distribution of VEGF between various organs. We sought to compare the basal VEGF protein expression in skeletal muscle, adipose tissue and mammary tumors from MMTV-PyMT mice and compared these levels against background nontransgenic FVB/N mice. Normal expression of VEGF protein was 69.4 ± 2.1 pg/mL in skeletal muscle and 59.0 ± 7.3 pg/mL in adipose tissue of wild-type (FVB background) control mice. The average expression level of VEGF protein in mammary tumors of MMTV-PyMT mice was 61.8 ± 31.3 pg/mL. Adipose tissue of MMTV-PyMT exhibited 324% greater (191.2 ± 37.6 pg/mL, p<0.01) VEGF protein expression compared to adipose tissue of control FVB mice, and 309% greater (p<0.05) VEGF protein compared to tumor VEGF expression. These data imply that stromal expression of VEGF may be a critical determinant regulating the angiogenic phenotype in breast cancer, and further suggests that cross-talk between the stromal environment and tumor is likely to be essential in promoting tumor growth and progression. NIH 5T32-[HL090610][1], AHA 10BGIA3630002, ACS 116837IRG0906101 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3269. doi:1538-7445.AM2012-3269 [1]: /lookup/external-ref?link_type=GEN&access_num=HL090610&atom=%2Fcanres%2F72%2F8_Supplement%2F3269.atom