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Effects of experimental type 1 diabetes and exercise training on angiogenic gene expression and capillarization in skeletal muscle

Riikka Kivelä^*,,1, Mika Silvennoinen^*,, Anna-Maria Touvra, T. Maarit Lehti^*, Heikki Kainulainen^*, and Veikko Vihko^*

^* LIKES Research Center for Sport and Health Sciences, Jyväskylä, Finland; and

Neuromuscular Research Center, Department of Biology of Physical Activity, University of Jyväskylä, Jyväskylä, Finland

¹Correspondence: LIKES Research Center, Rautpohjankatu 8a, Jyväskylä FIN-40740, Finland. E-mail: riikka.kivela{at}likes.fi

SPECIFIC AIMS

Angiogenesis is a process of new blood microvessel formation and it is regulated by pro- and antiangiogenic factors. Many physiological and pathological stimuli affect the expression of these factors. In diabetic skeletal muscles, angiogenesis has been shown to be impaired; whereas in healthy muscles, exercise increases the production of angiogenic growth factors and promotes the growth of new capillaries. The purpose of the present study was to investigate the changes in angiogenic gene expression and capillarization in diabetic skeletal muscle and to study whether exercise could reverse these changes.

PRINCIPAL FINDINGS

1. Streptozotocin-induced type 1 diabetes reduced the mRNA expression of angiogenic growth factors in mouse skeletal muscle
Diabetic and healthy animals were randomly assigned into 12 groups (n = 5 per group), which were either sedentary or trained for 1, 3, or 5 weeks. Training groups performed 1 h per day of treadmill running at 21 m/min and at an uphill incline of 2.5° for 5 days a week. Mice were killed 24 h after the last training session to focus on the effects of training and not the effects resulting from the last exercise bout. Oligonucleotide array analyses (Affymetrix Gene Chip MG U74Av2) and real-time PCR were performed to compare mRNA expression of angiogenesis-related genes in healthy and diabetic mice and the effects of exercise training. Diabetes decreased the mRNA levels of the major angiogenic growth factor, vascular endothelial growth factor (VEGF)-A, by 30–50% depending on time point (Fig. 1 ). The levels of VEGF-B, neuropilin-1, VEGF receptor-1 (Flt-1) and VEGF receptor-2 (Flk-1) were significantly reduced as well. Diabetes also down-regulated the expression of myoglobin and MnSOD.

View larger version (21K): [in this window] [in a new window]   Figure 1. Fold-change of VEGF-A, thrombospondin-1 (TSP-1), and connective tissue growth factor (CTGF) mRNA content in trained healthy (diagonally striped bars), sedentary diabetic (black bars) and trained diabetic (horizontally striped bars) mouse calf muscles compared to the sedentary healthy controls (white bars, set to value 1). Values are mean ± SE. *P < 0.05 vs. healthy sedentary controls, P < 0.05 vs. respective sedentary diabetic (=effect of training). Note the varying scale on the Y-axis due to the magnitude and direction of the changes.

2. Reduced VEGF-A protein content in diabetic muscles, but no change in VEGF-A, VEGF-B or VEGFR-2 protein localization
VEGF-A protein concentration in skeletal muscle homogenates was reduced in sedentary diabetic mice after 3 and 5 weeks of diabetes compared with the healthy sedentary mice (P < 0.05). In the endurance-trained diabetic groups, VEGF-A protein content was significantly decreased only after 5 weeks. After 3 weeks, trained diabetic animals tended to have higher VEGF-A content than sedentary diabetic mice (P = 0.086). We found no significant change in VEGF-A content in healthy trained mice compared with the healthy sedentary group.

3. The mRNA expression of angiogenesis inhibitors and of metallothioneins was increased by diabetes
The amount of thrombospondin-1 (TSP-1) mRNA, a known inhibitor of angiogenesis, was markedly increased in diabetic muscles (Fig. 1) as well as the concentration of retinoblastoma-like-2 (Rbl-2), which is considered a potent VEGF inhibitor. Diabetes also increased markedly the expression of metallothionein-1 and -2 at all time points. In diabetic muscles, Hif-1 mRNA levels were also higher than in healthy mice.

4. Increased mRNA expression of CNN family proteins
Extracellular matrix (ECM) remodeling is an important step in angiogenesis. The mRNA levels of matricellular proteins, connective tissue growth factor (CTGF), and cysteine-rich protein 61 (Cyr61) were increased in diabetic muscles (Fig. 1) . These proteins belong to the CNN protein family and are involved in ECM remodeling and angiogenesis.

5. Effects of exercise training on diabetes-induced changes in gene expression
Endurance training alleviated the diabetes-induced changes in the mRNA levels of VEGF-A, VEGF-B, neuropilin-1, myoglobin, MnSOD, metallothioneins, and Hif-1. However, exercise did not significantly decrease the levels of angiogenesis inhibitors TSP-1 or Rbl-2.

6. Decreased capillary-to-fiber ratio and muscle fiber cross-sectional area in diabetic mice
Both sedentary and trained diabetic mice showed a significant decrease in muscle fiber cross-sectional area (CSA) after 5 weeks compared with healthy controls (P < 0.05). Capillary density (cap – mm^–2) was not significantly changed due to diabetes, although it tended to be increased due to the reduced fiber cross-sectional area. Instead, the capillary-to-fiber ratio, which is commonly used to describe capillary supply to muscle fibers, was significantly decreased in both diabetic groups compared to the healthy control group (P < 0.05) but was not significantly different between the trained and sedentary groups in either diabetic or healthy mice.

DISCUSSION AND SIGNIFICANCE

In the present study we determined separate and combined effects of diabetes and exercise training on the expression of pro- and antiangiogenesis genes and proteins and on the capillarization in mouse skeletal muscle. The high blood glucose (Glc) concentration and considerable loss of body wt confirmed that streptozotocin had induced diabetes in the studied mice.

Diabetes decreased the mRNA concentration of many genes known to be involved in the regulation of angiogenesis, most interestingly those of VEGF-A and VEGF-B together with their receptors VEGFR-1/Flt1, VEGFR-2/Flk1, and neuropilin-1. The effect of diabetes on the VEGF-B and VEGF receptor mRNAs in skeletal muscle has not been reported earlier. In diabetic rabbits a reduction in VEGF-A mRNA levels in ischemic skeletal muscle has been observed previously. In cell cultures high Glc levels have been reported to inhibit VEGF-A production and insulin, in turn, to enhance it. In the present study, the amount of VEGF-A protein was also significantly reduced in sedentary diabetic muscles after 3 and 5 weeks of diabetes, which is in line with the mRNA results.

In addition to the mRNAs, we studied the localization of three important angiogenesis proteins—VEGF-A, VEGFR-2 and VEGF-B—in healthy and diabetic muscles. VEGF-A localized to myofibers and endothelial cells and VEGFR-2 to endothelial cells as has been shown earlier. To our knowledge, the localization of VEGF-B protein in skeletal muscle has not been reported earlier. The present findings showed that VEGF-B is localized mainly in larger blood vessels, capillaries, and interstitial cells. Similarly to VEGF-A, it was also detectable under the sarcolemma in a few muscle fibers. The present results confirm the presence of both VEGF-B mRNA and protein in skeletal muscle, and its localization to blood vessels supports its role in the maintenance of blood vessel endothelium. The localization of these three proteins was similar in healthy and diabetic skeletal muscle.

Diabetes also increased mRNA levels of several genes involved in angiogenesis. The most significant of these were the increases in TSP-1 and Rbl-2, both of which are known to inhibit angiogenesis. Increased expression of TSP-1 in the vessel wall of diabetic Zucker rats has been reported earlier, and it was proposed that it could be a direct response of vascular cells to Glc and, thus, a link between diabetes and atherosclerotic complications. The present results extend this finding from aorta to peripheral muscle tissues. Overexpression of retinoblastoma-like 2 (Rb2/Rbl2) decreased VEGF-A mRNA and protein levels in two tumor cell types, together with inhibited tumor angiogenesis, which suggest that Rbl2 is a potent VEGF-A inhibitor. In our experiment, diabetic muscles expressed higher mRNA levels of retinoblastoma-like 2 and lower mRNA levels of VEGF-A than healthy muscles.

Stress-inducible metallothionein-1 and -2 mRNA levels were more than 10-fold higher after the first week of diabetes. Thereafter, the levels slightly attenuated but remained still elevated throughout the whole experiment. The amounts of matricellular cysteine-rich proteins Cyr61, Cyr3, and connective tissue growth factor (CTGF) mRNAs were also greater in diabetic muscles. Cyr61 (CNN1) and CTGF (CNN2) are involved in ECM remodeling, and their proangiogenic activity suggests a role in the establishment and functioning of the vasculature. As a new finding based on the present results, it seems that, in addition to traditional angiogenic growth factors, diabetes could alter the mRNA expression of several ECM proteins, which are involved in the regulation of blood vessel growth.

VEGF-A, VEGF-B, myoglobin, and superoxide dismutase 2 mRNA levels were increased in trained diabetic mice compared with sedentary diabetic mice at least in some phase of the training period. In addition, diabetes-induced increases in Hif-1 and cysteine-rich proteins 3 and 61 were attenuated in the trained diabetic animals. Increases in VEGF-A, VEGF-B, and myoglobin mRNAs support enhanced capillary network and oxygen delivery to muscle fibers. Training also slightly reduced the diabetes-induced expression of metallothioneins but also increased their expression after the first week of training in healthy animals.

In line with the reduced levels of VEGF-A and other proangiogenic factors, and the increased levels of angiogenesis inhibitors, capillary-to-fiber ratio was lower in diabetic mice compared with healthy controls. Exercise training could not restore capillarization in diabetic mice, and the training-induced increase was not statistically significant in healthy mice either.

In conclusion, the present study showed that streptozotocin-induced diabetes and the subsequent hyperglycemia reduce the mRNA levels of proangiogenic proteins and increase those of antiangiogenic ones, together with decreased capillarization. This change of balance may be one of the major reasons for the markedly increased risk for peripheral cardiovascular complications in diabetes. Endurance training alleviated some of these changes but did not fully restore the diabetes-induced defects. These training effects, seen in the mRNA levels of angiogenesis-related genes, may be one of the mechanisms responsible for the beneficial effects of regular endurance exercise in diabetic patients. These data suggest that reduced skeletal muscle capillarization in type 1 diabetes is associated with the dysregulation of complex angiogenesis pathways (Fig. 2 ).

View larger version (16K): [in this window] [in a new window]   Figure 2. A schematic summary based on the existing literature and the findings of the present study. Type 1 diabetes impairs skeletal muscle capillarization and/or angiogenesis by reducing the mRNA concentration of pro-angiogenic factors and increasing the expression of antiangiogenic factors. Exercise training induces opposite effect on pro-angiogenic factors, which in the present experimental model did not completely compensate the diabetes-induced changes. Effects of exercise training on antiangiogenic factors have not been studied earlier and in the present experiment it could not reverse the increased expression in diabetic muscles (dashed line). In the figure, increased expression or activation is marked with + and decreased expression or inhibition with –.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4780fje

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