Parallels between wound healing, epimorphic regeneration and solid tumors.

Striking similarities between wound healing, epimorphic regeneration and the progression of solid tumors have been uncovered by recent studies. In this Review, we discuss systemic effects of tumorigenesis that are now being appreciated in epimorphic regeneration, including genetic, cellular and metabolic heterogeneity, changes in circulating factors, and the complex roles of immune cells and immune modulation at systemic and local levels. We suggest that certain mechanisms enabling regeneration may be co-opted by cancer to promote growth at primary and metastatic sites. Finally, we advocate that working with a unified approach could complement research in both fields.

Systemic cell cycle activation is induced following complex tissue injury in axolotl.

Activation of progenitor cells is crucial to promote tissue repair following injury in adult animals. In the context of successful limb regeneration following amputation, progenitor cells residing within the stump must re-enter the cell cycle to promote regrowth of the missing limb. We demonstrate that in axolotls, amputation is sufficient to induce cell-cycle activation in both the amputated limb and the intact, uninjured contralateral limb. Activated cells were found throughout all major tissue populations of the intact contralateral limb, with internal cellular populations (bone and soft tissue) the most affected. Further, activated cells were additionally found within the heart, liver, and spinal cord, suggesting that amputation induces a common global activation signal throughout the body. Among two other injury models, limb crush and skin excisional wound, only limb crush injuries were capable of inducing cellular responses in contralateral uninjured limbs but did not achieve activation levels seen following limb loss. We found this systemic activation response to injury is independent of formation of a wound epidermis over the amputation plane, suggesting that injury-induced signals alone can promote cellular activation. In mammals, mTOR signaling has been shown to promote activation of quiescent cells following injury, and we confirmed a subset of activated contralateral cells is positive for mTOR signaling within axolotl limbs. These findings suggest that conservation of an early systemic response to injury exists between mammals and axolotls, and propose that a distinguishing feature in species capable of full regeneration is converting this initial activation into sustained and productive growth at the site of regeneration.

Folate depletion induces erythroid differentiation through perturbation of de novo purine synthesis.

Folate, an essential vitamin, is a one-carbon acceptor and donor in key metabolic reactions. Erythroid cells harbor a unique sensitivity to folate deprivation, as revealed by the primary pathological manifestation of nutritional folate deprivation: megaloblastic anemia. To study this metabolic sensitivity, we applied mild folate depletion to human and mouse erythroid cell lines and primary murine erythroid progenitors. We show that folate depletion induces early blockade of purine synthesis and accumulation of the purine synthesis intermediate and signaling molecule, 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR), followed by enhanced heme metabolism, hemoglobin synthesis, and erythroid differentiation. This is phenocopied by inhibition of folate metabolism using the inhibitor SHIN1, and by AICAR supplementation. Mechanistically, the metabolically driven differentiation is independent of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine 5'-monophosphate-activated protein kinase (AMPK) and is instead mediated by protein kinase C. Our findings suggest that folate deprivation-induced premature differentiation of erythroid progenitor cells is a molecular etiology to folate deficiency-induced anemia.

Cycloguanil and Analogues Potently Target DHFR in Cancer Cells to Elicit Anti-Cancer Activity.

Dihydrofolate reductase (DHFR) is an established anti-cancer drug target whose inhibition disrupts folate metabolism and STAT3-dependent gene expression. Cycloguanil was proposed as a DHFR inhibitor in the 1950s and is the active metabolite of clinically approved plasmodium DHFR inhibitor Proguanil. The Cycloguanil scaffold was explored to generate potential cancer therapies in the 1970s. Herein, current computational and chemical biology techniques were employed to re-investigate the anti-cancer activity of Cycloguanil and related compounds. In silico modeling was employed to identify promising Cycloguanil analogues from NCI databases, which were cross-referenced with NCI-60 Human Tumor Cell Line Screening data. Using target engagement assays, it was found that these compounds engage DHFR in cells at sub-nanomolar concentrations; however, growth impairments were not observed until higher concentrations. Folinic acid treatment rescues the viability impairments induced by some, but not all, Cycloguanil analogues, suggesting these compounds may have additional targets. Cycloguanil and its most promising analogue, NSC127159, induced similar metabolite profiles compared to established DHFR inhibitors Methotrexate and Pyrimethamine while also blocking downstream signaling, including STAT3 transcriptional activity. These data confirm that Cycloguanil and its analogues are potent inhibitors of human DHFR, and their anti-cancer activity may be worth further investigation.

Transcriptomic landscape of the blastema niche in regenerating adult axolotl limbs at single-cell resolution.

Regeneration of complex multi-tissue structures, such as limbs, requires the coordinated effort of multiple cell types. In axolotl limb regeneration, the wound epidermis and blastema have been extensively studied via histology, grafting, and bulk-tissue RNA-sequencing. However, defining the contributions of these tissues is hindered due to limited information regarding the molecular identity of the cell types in regenerating limbs. Here we report unbiased single-cell RNA-sequencing on over 25,000 cells from axolotl limbs and identify a plethora of cellular diversity within epidermal, mesenchymal, and hematopoietic lineages in homeostatic and regenerating limbs. We identify regeneration-induced genes, develop putative trajectories for blastema cell differentiation, and propose the molecular identity of fibroblast-like blastema progenitor cells. This work will enable application of molecular techniques to assess the contribution of these populations to limb regeneration. Overall, these data allow for establishment of a putative framework for adult axolotl limb regeneration.

Identification of regenerative roadblocks via repeat deployment of limb regeneration in axolotls.

Axolotl salamanders are powerful models for understanding how regeneration of complex body parts can be achieved, whereas mammals are severely limited in this ability. Factors that promote normal axolotl regeneration can be examined in mammals to determine if they exhibit altered activity in this context. Furthermore, factors prohibiting axolotl regeneration can offer key insight into the mechanisms present in regeneration-incompetent species. We sought to determine if we could experimentally compromise the axolotl's ability to regenerate limbs and, if so, discover the molecular changes that might underlie their inability to regenerate. We found that repeated limb amputation severely compromised axolotls' ability to initiate limb regeneration. Using RNA-seq, we observed that a majority of differentially expressed transcripts were hyperactivated in limbs compromised by repeated amputation, suggesting that mis-regulation of these genes antagonizes regeneration. To confirm our findings, we additionally assayed the role of amphiregulin, an EGF-like ligand, which is aberrantly upregulated in compromised animals. During normal limb regeneration, amphiregulin is expressed by the early wound epidermis, and mis-expressing this factor lead to thickened wound epithelium, delayed initiation of regeneration, and severe regenerative defects. Collectively, our results suggest that repeatedly amputated limbs may undergo a persistent wound healing response, which interferes with their ability to initiate the regenerative program. These findings have important implications for human regenerative medicine.

Maternal cardiovascular changes during pregnancy and postpartum in mice.

Genetically altered mice may provide useful models for exploring cardiovascular regulation during pregnancy and postpartum if changes in mice mimic humans. We found in awake ICR (CD-1) mice at 17.5 days gestation that hematocrit was reduced 18%, and the pressor response to intravenous angiotensin II was reduced ~33%. Arterial pressure in awake mice was 12% lower in early pregnancy (3.5 days) than late pregnancy (17.5 days) and postpartum (3 and 17 days after delivery), whereas heart rate was 10-20% higher in the peripartum period (17.5 days gestation and 3 days postpartum). In late pregnancy, cardiac output under isoflurane anesthesia was 64% higher than in nonpregnant mice, due to a 37% increase in stroke volume and a 17% increase in heart rate. All changes P < 0.05. We conclude that, as in humans, mice exhibit hypotension in early pregnancy, and a blunted pressor response to angiotensin II, a decrease in hematocrit, and a marked increase in cardiac output in late pregnancy.