Aaron Avivi

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 in muscles used in digging and in other energetic activities, resulting in a shorter diffusion distance for oxygen. Vascular endothelial growth factor (VEGF) is an angiogenic factor that is critical for angiogenesis during development and is found in response to tissue ischemia. We demonstrate here that due to physiological differences, the Spalax muscle regulatory mechanism for VEGF is different than in Rattus muscle. In vivo, the constitutive level of the VEGF mRNA and the mRNA levels of its transcriptional regulator HIF‐1α and its mRNA stabilizer HuR are significantly higher in Spalax muscle than in Rattus muscle. Furthermore, as opposed to Rattus , the mRNA levels of HIF‐1α, HuR, VEGF , as well as that of LDH‐A , the enzyme that catalyzes the production of lactate, an accepted marker of anaerobic metabolism, are not increased in Spalax after hypoxia. However, ex vivo, when oxygenation by blood vessels is no longer relevant, the expression pattern of all these genes is similar in the two rodents under both normoxic and hypoxic conditions. Our studies provide evidence that the highly vascularized muscle in Spalax , the most energy consuming tissue during digging, is resistant to the effects of oxygen deprivation. The significance of these results with respect to ischemic vascular disease is abundantly clear.

P53 is a transcription factor involved in maintaining genomic integrity by regulating genes involved in cell cycle arrest, DNA repair, and programmed cell death. Various post transcriptional modifications result in activated p53 with varying binding affinity to its targets. The other members of the p53 family (p63, p73) and associated proteins also contribute to the specificity of gene activation resulting in the final cell responses. p53 is commonly mutated in human cancer and is activated by diverse cellular events, including hypoxia. Many sources of genetic diversity, including random or stress-related mutagenesis, affect normal species evolution. The blind subterranean mole rat lives in sealed underground tunnels, subjected to routine hypoxia due to abrupt and sharp changes in O2 supply. We cloned the mole rat's p53 gene and identified two amino acid substitutions in its binding domain, in the same positions that are mutated in cancer. These substitutions lead to increased p53 activation of DNA-repair...

Blind subterranean mole rats of the Spalax ehrenbergi superspecies in Israel have evolved multiple adaptive strategies to face underground hypoxia. Hypoxia‐inducible factor‐1α (HIF‐1α) and erythropoietin (Epo) are key factors in the development of normal erythropoiesis and angiogenesis. Here, we demonstrate via real‐time polymerase chain reaction (PCR) quantification that Spalax fetal liver and kidney express higher levels of Epo mRNA than Rattus , generating reinforcement of fetal erythropoiesis underground and adapting it to life underground in an atmosphere of abrupt and sharp fluctuations of O ₂ supply. In neonates, Rattus liver and kidney express higher Epo levels than Spalax under both normoxia and hypoxia, probably due to Rattus ineffective erythropoiesis during embryonic life and its birth in a poorly ventilated breeding nest under ground. Adult Rattus kidney and liver, and adult Spalax liver express similar levels of Epo mRNA under normoxia and hypoxia. However, adult Spalax hypoxic kidney, the major site of erythropoietin production in adult mammals, shows levels that were twice as high as that of Rattus. Spalax expresses remarkably higher levels of HIF‐1α mRNA than Rattus at all developmental stages studied, which peaked in neonates, as an adaptation against hypoxia.

The tumor suppressor genep53controls cellular response to a variety of stress conditions, including DNA damage and hypoxia, leading to growth arrest and/or apoptosis. Inactivation ofp53, found in 40–50% of human cancers, confers selective advantage under hypoxic microenvironment during tumor progression. The mole rat,Spalax, spends its entire life cycle underground at decidedly lower oxygen tensions than any other mammal studied. Because a wide range of respiratory adaptations to hypoxic stress evolved inSpalax, we speculated that it might also have developed hypoxia adaptation mechanisms analogous to the genetic/epigenetic alterations acquired during tumor progression. ComparingSpalaxwith human and mouse p53 revealed an arginine (R) to lysine (K) substitution inSpalax(Arg-174 in human) in the DNA-binding domain, identical to known tumor associated mutations. Multiple p53 sequence alignments with 41 additional species confirmed that Arg-174 is highly conserved. Reporter assays uncovered thatSpalaxp53 protein is unable to induce apoptosis-regulating target genes, resulting in no expression ofapaf1and partial expression ofpuma, pten, andnoxa. However, cell cycle arrest and p53 stabilization/homeostasis genes were overactivated bySpalaxp53. Lys-174 was found critical forapaf1expression inactivation. A DNA-free p53 structure model predicts that Arg-174 is important for dimerization, whereasSpalaxLys-174 prevents such interactions. Similar neighboring mutations found in human tumors favor growth arrest rather than apoptosis. We hypothesize that, in an analogy with human tumor progression,Spalaxunderwent remarkable adaptive p53 evolution during 40 million years of underground hypoxic life.

Blind subterranean mole rats (Spalax, Spalacidae) evolved adaptive strategies to cope with hypoxia that climaxes during winter floods in their burrows. By using real-time PCR, we compared gene expression oferythropoietin(Epo), a key regulator of circulating erythrocytes, andhypoxia-inducible factor 1α (HIF-1α),Epoexpression inducer, in the kidneys ofSpalaxand white rats,Rattus norvegicus. Our results show significantly higher, quicker, and longer responses to different O₂levels inSpalaxcompared withRattus. (i) In normoxia, bothSpalaxandRattuskidneys produce small amounts ofEpo. Maximal expression ofRattus Epois noticed after a 4-h hypoxia at 6% O₂. Under these conditions,Spalax Epolevels are 3-fold higher than inRattus. After 24 h of 10% O₂,Spalax Eporeaches its maximal expression, remarkably 6-fold higher than the maximum inRattus; (ii) theHIF-1α level in normoxia is 2-fold higher inSpalaxthan inRattus. Spalax HIF-1α achieves maximal expression after 4-h hypoxia at 3% O₂, a 2-fold increase compared with normoxia, whereas no significant change was detected inRattus HIF-1α at any of the conditions studied; (iii) at 6% O₂for 10 h, in whichRattuscannot survive,EpoandHIF-1α levels inSpalax galili, living in heavily flooded soils, are higher than inSpalax judaei, residing in light aerated soil. We suggest that this pattern ofEpoandHIF-1α expression is a substantial contribution to the adaptive strategy of hypoxia tolerance inSpalax, evolved during 40 million years of evolution to cope with underground hypoxic stress.

The blind subterranean mole rat superspecies Spalax ehrenbergi is an extreme example of mammalian adaptation to life underground. Though this rodent is totally visually blind, harboring a drastically degenerated subcutaneous rudimentary eye, its daily activity rhythm is entrainable to LD cycles. This indicates that it confers light information to the clock, as has been previously shown by the authors in behavioral studies as well as by molecular analyses of its Clock/MOP3 and its three Per genes. The Cryptochrome (Cry) genes found in animals and plants act both as photoreceptors and as essential components of the negative feedback mechanism of the biological clock. To further understand the circadian system of this unique mammal, the authors cloned and characterized the open reading frame of Spalax Cry1 and Cry2. The Spalax CRY1 protein is significantly closer to the human homolog than to the mice one, in contrast to the evolutionary expectations. They have found two isoforms of Cry2 in Spalax, which differ in their 5’ end of the open reading frame and defined their expression in Spalax populations. They found a large and significant excess of heterozygotes of sCry2 (sCry2L/S genotype). Both sCry1 and sCry2 mRNAs were found in the SCN, the eye, the harderian gland, as well as in a wide range of peripheral tissues. Their expression pattern under different LD conditions has also been analyzed. As was already shown for other circadian genes, despite being blind and living in darkness, the Cry genes of Spalax behave in a similar, though not identical, pattern as in sighted animals. Once again, the results indicate that the uniquely hypertrophied harderian gland of Spalax plays a key role in its circadian system.

Angiogenic therapy with vascular endothelial growth factor (VEGF) has been proposed as a treatment paradigm for patients suffering from an insufficiency of collateral vessels. Diabetes is associated with increase in the production of VEGF and therefore additional VEGF may not be beneficial. Accordingly, we sought to determine the efficacy of VEGF therapy to augment collateral formation and tissue perfusion in a diabetic mouse ischemic hindlimb model. Diabetic and non-diabetic mice were studied in parallel for the efficacy of VEGF administration. Diabetes was induced with streptozotocin. Hindlimb ischemia was produced by severing the left iliac artery. An outlet tube from an osmotic infusion pump with placebo/ 500 micrograms of plasmid-DNA encoding VEGF was fenestrated and tunneled into the left quadriceps muscle. VEGF induced more rapid and complete restoration of blood flow in normal mice. However, in the setting of diabetes there was no difference between VEGF Vs. placebo in the rate or adequacy of flow restoration. There was a significant increase in smooth muscle actin and Factor-VIII antigen densities in diabetic animals and in animals which received VEGF. Angiogenic therapy with VEGF in the setting of diabetes does not appear to have the beneficial effects seen in the absence of diabetes.

We demonstrated that a subterranean, visually blind mammal has a functional set of threePergenes that are important components of the circadian clockwork in mammals. The mole rat superspeciesSpalax ehrenbergiis a blind subterranean animal that lives its entire life underground in darkness. It has degenerated eyes, but the retina and highly hypertrophic harderian gland are involved in photoperiodic perception. All threePergenes oscillate with a periodicity of 24 h in the suprachiasmatic nuclei, eye, and harderian gland and are expressed in peripheral organs. This oscillation is maintained under constant conditions. The light inducibility ofsPer1andsPer2, which are similar in structure to those of other mammals, indicates the role of these genes in clock resetting. However,sPer3is unique in mammals and has two truncated isoforms, and its expressional analysis leaves its function unresolved.Per's expression analysis in the harderian gland suggests an important participation of this organ in the stabilization and resetting mechanism of the central pacemaker in the suprachiasmatic nuclei and in unique adaptation to life underground.