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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 13, 2005 as doi:10.1096/fj.04-3414fje. |
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Laboratory of Animals Molecular Evolution, Institute of Evolution, University of Haifa, Mt. Carmel, Haifa 31905, Israel; and
* Technion Faculty of Medicine, Haifa, Israel
1Correspondence: Laboratory of Animals Molecular Evolution, Institute of Evolution, University of Haifa, Mt. Carmel, Haifa 31905, Israel. E-mail: aaron{at}esti.haifa.ac.il
SPECIFIC AIMS
The blind subterranean mole rat superspecies Spalax ehrenbergi has evolved adaptations that allow it to survive and carry out intensive activities in its highly hypoxic underground sealed burrows. A key component of this adaptation is a higher capillary density in some Spalax tissues, primarily muscles involved in digging and energetic activities, resulting in a shorter diffusion distance for oxygen. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that is critical in the ability to grow new blood vessels for physiological processes, such as embryogenesis, and in pathological processes such as tumor growth and metastasis. One of the key factors controlling VEGF expression is oxygen tension. The lack of oxygen (hypoxia) induces VEGF expression at the levels of mRNA transcription and stabilization.
In this study we sought to explain the enigma of why Spalax muscle does not increase VEGF with hypoxia as reported by our group previously. Using real-time quantitative PCR, mRNA expression of VEGF, HIF-1
, its transcription factor, and HuR, its mRNA stabilizer, were compared in skeletal muscles of Spalax and Rattus under normoxic and hypoxic conditions, in vivo and ex vivo. We also examined the production of lactate, an accepted marker of anaerobic metabolism, as well as the expression of lactate dehydrogenase A, the enzyme that catalyzes the production of lactate, in response to hypoxia in the muscles of both species.
PRINCIPAL FINDINGS
1. In vivo measurements of VEGF, HIF-1, and HuR mRNAs in Spalax and Rattus muscles under ambient conditions
Spalax and Rattus VEGF, HIF-1, and HuR mRNAs extracted from animals kept under normoxic conditions (21% O2), were quantitated. Spalax constitutive levels of all three mRNAs are significantly higher than in Rattus. VEGF mRNA is 1.6 ± 0.2 (P<0.05) -fold higher in Spalax compared with Rattus, HIF-1 mRNA is 4.2 ± 0.35 (P<0.05) -fold higher in Spalax compared with Rattus, and HuR mRNA is 23.4 ± 0.12 (P<0.01) -fold higher in Spalax compared with Rattus.
2. Determination of the in vivo induction of VEGF, HIF-1, and HuR mRNAs in Spalax and Rattus muscles under conditions of hypoxia
We exposed live animals of Spalax and Rattus to controlled 6% O2 for 4 h and quantitated mRNA. As shown in Fig. 1
, we found that the mRNA of all three genes was significantly increased in Rattus muscle as a result of hypoxic stress. Rattus VEGF was increased by 2.2 ± 0.3 (P<0.05) times; Rattus HIF-1 was increased by 2.83 ± 0.48 (P<0.05) times; and Rattus HuR increased by 2.24 ± 0.1 (P<0.01) times. In hypoxia-exposed Spalax muscles, however, we observed a small but significant decrease in VEGF (0.7±0.1; P<0.05) and HuR (0.8±0.03; P<0.01) and no change in HIF-1 (1.03±0.03; P<0.01).
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3. In vivo expression of lactate and LDH-A mRNA in Spalax and Rattus muscles
We found that, under in vivo conditions, in Rattus muscles there was a marked increase (1.4±0.2; P<0.05) in lactate levels in response to hypoxia whereas in Spalax muscles there was no such-increase (0.8±0.1; P<0.05). Moreover, expression of LDH-A mRNA, the enzyme that catalyzes production of lactate, is also significantly up-regulated in Rattus muscles in response to hypoxia (1.9±0.4; P<0.05), but there was no increase in the Spalax muscles under similar conditions (0.87±0.13; P<0.05)
4. Ex vivo expression of VEGF, HIF-1, and HuR mRNAs in Spalax and Rattus muscles
We removed a neck muscle tissue from Spalax and Rattus, manually minced the tissue, and incubated it for 18 h under normoxic conditions (21% O2) and at 1% O2 hypoxia. As demonstrated in Fig. 2
, ex vivo results are different from in vivo. In this experiment there was no difference in the hypoxic/normoxic expression ratio of these three genes in Spalax and Rattus. Both species exhibited a similar and significant increase in muscle mRNA expression under hypoxia. VEGF increased by 2.9±0.5 (P<0.05) times in Spalax and by 2.4 ± 0.2 (P<0.05) in Rattus. HIF-1 was increased by 3.43 ± 0.28 (P<0.05) in Spalax and by 3.27 ± 017 (P<0.01) in Rattus. HuR increased by 3.44 ± 1.02 (P<0.05) in Spalax and by 3.35 ± 0.9 (P<0.05) in Rattus.
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CONCLUSIONS AND SIGNIFICANCE
All higher organisms have developed adaptive mechanisms to detect and respond to hypoxia. However, animals that chronically inhabit hypoxic ecological niches developed uniquely effective strategies and adaptations to survive under hypoxia. Nevertheless, Spalax differs dramatically from all diving mammals that are exposed intermittently to hypoxia, recharging O2 breathing, and from high-altitude mammals that are often limited in their vertical ascent and hypoxic exposure. Spalax, by contrast, spends all its life underground in sealed burrows and experiences hypoxia down to 7% O2, and probably much lower when the burrows are flooded by rainwater. Consequently, Spalax is confronting hypoxia due to sharp and abrupt fluctuations of oxygen supply, primarily during its winter activities. Permanent activity under hypoxia necessitates constant O2 delivery to tissues, especially muscles used in burrowing. Spalax achieves this essentially by decreasing the diffusion distance for oxygen in the skeletal muscle tissue by increasing the capillary density, which has been demonstrated to be critically dependent on the angiogenic factor VEGF. VEGF transcription, as well as that of several other hypoxia-inducible genes, is increased with hypoxia via binding of HIF-1. Nevertheless, the half-life of VEGF mRNA is very short, therefore limiting the amount of its gene product. HuR, a RNA binding protein that belongs to the embryonic lethal abnormal visual (ELAV) protein family, is involved in increasing VEGF mRNA stability under hypoxic stress.
We have shown that, under in vivo conditions, VEGF was constitutively elevated in Spalax muscle at levels, which were significantly higher than in Rattus muscle. The higher constitutive VEGF mRNA expression in Spalax muscle was correlated with a significantly higher blood vessel concentration compared with Rattus muscle. Exposure of Rattus and Spalax to a hypoxic environment revealed further differences between the two species. In Rattus and in those Spalax tissues where the blood vessel concentration was similar to Rattus tissues, we found that VEGF was increased with hypoxia to a similar degree. However, in Spalax muscle we found that VEGF was not increased with hypoxia while under the same environmental conditions it was increased in Rattus muscle.
To understand the molecular basis of the differences in the behavior of muscles between Spalax and Rattus, we studied the expression of VEGF and the genes that regulate its transcription, HIF1, and its mRNA stabilization, HuR, under both in vivo conditions, where oxygenation of the animal tissues is supported by its network of blood vessels, and ex vivo when blood vessel density is irrelevant for tissue oxygenation and totally dependent on diffusion from the experimental milieu.
Our finding demonstrates that under in vivo conditions, in an ambient atmosphere, not only VEGF, but also HIF-1, the hypoxia-inducible subunit of HIF-1, and HuR are expressed at higher levels in Spalax muscles compared with Rattus muscles. As the steady-state level of VEGF mRNA is dependent on HIF-1 and HuR, it is logical that the higher constitutive level of VEGF mRNA is supported by the higher constitutive levels of HIF-1 and HuR. Furthermore, hypoxic stress, in vivo, up-regulates all three genes in Rattus significantly, but there is no hypoxia-mediated increase of any of these genes in the Spalax muscles.
We may explain the in vivo differences of these genes expression in the muscles as being due to physiological differences in Spalax muscles vs. Rattus muscles. We propose that in vivo when the animals are exposed to low oxygen tension, the Spalax muscles do not get hypoxic due to the high concentration of blood vessels; therefore, VEGF and its regulators do not increase whereas the Rattus muscles get hypoxic and react accordingly by up-regulation of the expression of these genes. This hypothesis is supported by our lactate and LDH-A mRNA measurements in normoxic and hypoxic muscles of both rodents. Lactate dehydrogenase catalyzes the interconversion of pyruvate and lactate. In anaerobic cells conversion of pyruvate to lactate is essential for continued glycolytic flux. The LDH-A gene encodes the isoform that is adapted for the anaerobic function, and is the predominant form expressed in glycolytically active skeletal muscle. Moreover, expression of LDH-A is inducible by hypoxia and HIF-1 is critical to the mechanism of its hypoxic induction. We demonstrate here that lactate production, an accepted marker of anaerobic metabolism, is markedly increased in Rattus muscles in response to hypoxia whereas in Spalax there was no such increase. Similarly, we show that LDH-A mRNA expression was up-regulated in the Rattus muscle under hypoxia while there was no such increase in Spalax muscle. That this is physiological is demonstrated in our ex vivo experiments. Thus, when the blood vessel density becomes irrelevant (ex vivo) and the mechanism of oxygenation is the same in Spalax and Rattus, depending on diffusion of oxygen from the culture media, both rodents up-regulated VEGF, HIF-1, and HuR to the same degree with hypoxia.
These studies provide direct evidence that when a tissue is sufficiently vascularized, as in Spalax, its resistance to the effects of oxygen deprivation is markedly increased. As our ability to modulate tissue vascularity becomes a reality, the significance of our results is apparent with respect to ischemic vascular disease and protection of tissues from environmentally imposed hypoxic stress.
Functional genomic studies of comparative physiology of hypoxia are suggested as a potential approach to understand the genetic basis of the physiological processes and evolutionary adaptations of wild and domestic animals. Future studies in our laboratory aim at probing the extensive adaptive battery of genetic responses of Spalax to variable hypoxic stress through comprehensive functional genomic studies that will unravel the numerous genes cooperating in Spalax hypoxia tolerance. This knowledge could later be used in human gene therapy in the fight against ischemia and cancer and may contribute to a better understanding of life in extreme environments.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3414fje;
This article has been cited by other articles:
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