A Pro-Regenerative Supramolecular Prodrug Protects Against and Repairs Colon Damage in Experimental Colitis.

Structural repair of the intestinal epithelium is strongly correlated with disease remission in inflammatory bowel disease (IBD); however, ulcer healing is not addressed by existing therapies. To address this need, this study reports the use of a small molecule prolyl hydroxylase (PHD) inhibitor (DPCA) to upregulate hypoxia-inducible factor one-alpha (HIF-1α) and induce mammalian regeneration. Sustained delivery of DPCA is achieved through subcutaneous injections of a supramolecular hydrogel, formed through the self-assembly of PEG-DPCA conjugates. Pre-treatment of mice with PEG-DPCA is shown to protect mice from epithelial erosion and symptoms of dextran sodium sulfate (DSS)-induced colitis. Surprisingly, a single subcutaneous dose of PEG-DPCA, administered after disease onset, leads to accelerated weight gain and complete restoration of healthy tissue architecture in colitic mice. Rapid DPCA-induced restoration of the intestinal barrier is likely orchestrated by increased expression of HIF-1α and associated targets leading to an epithelial-to-mesenchymal transition. Further investigation of DPCA as a potential adjunctive or stand-alone restorative treatment to combat active IBD is warranted.

Epithelial-mesenchymal transition: an organizing principle of mammalian regeneration.

Introduction: The MRL mouse strain is one of the few examples of a mammal capable of healing appendage wounds by regeneration, a process that begins with the formation of a blastema, a structure containing de-differentiating mesenchymal cells. HIF-1α expression in the nascent MRL wound site blastema is one of the earliest identified events and is sufficient to initiate the complete regenerative program. However, HIF-1α regulates many cellular processes modulating the expression of hundreds of genes. A later signal event is the absence of a functional G1 checkpoint, leading to G2 cell cycle arrest with increased cellular DNA but little cell division observed in the blastema. This lack of mitosis in MRL blastema cells is also a hallmark of regeneration in classical invertebrate and vertebrate regenerators such as planaria, hydra, and newt. Results and discussion: Here, we explore the cellular events occurring between HIF-1α upregulation and its regulation of the genes involved in G2 arrest (EVI-5, γH3, Wnt5a, and ROR2), and identify epithelial-mesenchymal transition (EMT) (Twist and Slug) and chromatin remodeling (EZH-2 and H3K27me3) as key intermediary processes. The locus of these cellular events is highly regionalized within the blastema, occurring in the same cells as determined by double staining by immunohistochemistry and FACS analysis, and appears as EMT and chromatin remodeling, followed by G2 arrest determined by kinetic expression studies.

Prolyl-hydroxylase inhibitor-induced regeneration of alveolar bone and soft tissue in a mouse model of periodontitis through metabolic reprogramming.

Bone injuries and fractures reliably heal through a process of regeneration with restoration to original structure and function when the gap between adjacent sides of a fracture site is small. However, when there is significant volumetric loss of bone, bone regeneration usually does not occur. In the present studies, we explore a particular case of volumetric bone loss in a mouse model of human periodontal disease (PD) in which alveolar bone surrounding teeth is permanently lost and not replaced. This model employs the placement a ligature around the upper second molar for 10 days leading to inflammation and bone breakdown and faithfully replicates the bacterially-induced inflammatory etiology of human PD to induce bone degeneration. After ligature removal, mice are treated with a timed-release formulation of a small molecule inhibitor of prolylhydroxylases (PHDi; 1,4-DPCA) previously shown to induce epimorphic regeneration of soft tissue in non-regenerating mice. This PHDi induces high expression of HIF-1α and is able to shift the metabolic state from OXPHOS to aerobic glycolysis, an energetic state used by stem cells and embryonic tissue. This regenerative response was completely blocked by siHIF1a. In these studies, we show that timed-release 1,4-DPCA rapidly and completely restores PD-affected bone and soft tissue with normal anatomic fidelity and with increased stem cell markers due to site-specific stem cell migration and/or de-differentiation of local tissue, periodontal ligament (PDL) cell proliferation, and increased vascularization. In-vitro studies using gingival tissue show that 1,4-DPCA indeed induces de-differentiation and the expression of stem cell markers but does not exclude the role of migrating stem cells. Evidence of metabolic reprogramming is seen by the expression of not only HIF-1a, its gene targets, and resultant de-differentiation markers, but also the metabolic genes Glut-1, Gapdh, Pdk1, Pgk1 and Ldh-a in jaw periodontal tissue.

Unlocking mammalian regeneration through hypoxia inducible factor one alpha signaling.

Historically, the field of regenerative medicine has aimed to heal damaged tissue through the use of biomaterials scaffolds or delivery of foreign progenitor cells. Despite 30 years of research, however, translation and commercialization of these techniques has been limited. To enable mammalian regeneration, a more practical approach may instead be to develop therapies that evoke endogenous processes reminiscent of those seen in innate regenerators. Recently, investigations into tadpole tail regrowth, zebrafish limb restoration, and the super-healing Murphy Roths Large (MRL) mouse strain, have identified ancient oxygen-sensing pathways as a possible target to achieve this goal. Specifically, upregulation of the transcription factor, hypoxia-inducible factor one alpha (HIF-1α) has been shown to modulate cell metabolism and plasticity, as well as inflammation and tissue remodeling, possibly priming injuries for regeneration. Since HIF-1α signaling is conserved across species, environmental or pharmacological manipulation of oxygen-dependent pathways may elicit a regenerative response in non-healing mammals. In this review, we will explore the emerging role of HIF-1α in mammalian healing and regeneration, as well as attempts to modulate protein stability through hyperbaric oxygen treatment, intermittent hypoxia therapy, and pharmacological targeting. We believe that these therapies could breathe new life into the field of regenerative medicine.

An injectable hydrogel-formulated inhibitor of prolyl-4-hydroxylase promotes T regulatory cell recruitment and enhances alveolar bone regeneration during resolution of experimental periodontitis.

The hypoxia-inducible factor 1α (HIF-1α) is critically involved in tissue regeneration. Hence, the pharmacological prevention of HIF-1α degradation by prolyl hydroxylase (PHD) under normoxic conditions is emerging as a promising option in regenerative medicine. Using a mouse model of ligature-induced periodontitis and resolution, we tested the ability of an injectable hydrogel-formulated PHD inhibitor, 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA/hydrogel), to promote regeneration of alveolar bone lost owing to experimental periodontitis. Mice injected subcutaneously with 1,4-DPCA/hydrogel at the onset of periodontitis resolution displayed significantly increased gingival HIF-1α protein levels and bone regeneration, as compared to mice treated with vehicle control. The 1,4-DPCA/hydrogel-induced increase in bone regeneration was associated with elevated expression of osteogenic genes, decreased expression of pro-inflammatory cytokine genes, and increased abundance of FOXP3+ T regulatory (Treg) cells in the periodontal tissue. The enhancing effect of 1,4-DPCA/hydrogel on Treg cell accumulation and bone regeneration was reversed by AMD3100, an antagonist of the chemokine receptor CXCR4 that mediates Treg cell recruitment. In conclusion, the administration of 1,4-DPCA/hydrogel at the onset of periodontitis resolution promotes CXCR4-dependent accumulation of Treg cells and alveolar bone regeneration, suggesting a novel approach for regaining bone lost due to periodontitis.

Supramolecular Polymer Hydrogels for Drug-Induced Tissue Regeneration.

Supramolecular polymers self-assemble into nanofibers, micelles, and other nanostructures through weak noncovalent interactions between subunits. Such systems possess attractive properties for use in a variety of practical settings such as energy, sustainability, and healthcare. In regenerative medicine, a common approach involves implanting a supramolecular material containing cell and growth factor binding motifs directly into a diseased or traumatized tissue defect, whereupon it interacts with and/or recruits components of the biological system to induce tissue healing. Here we introduce a supramolecular therapeutic in which tissue regeneration is orchestrated by a supramolecular polymer prodrug implanted subcutaneously in a remote tissue. Our approach exploits a hydrophobic small-molecule inhibitor of prolyl hydroxylase enzyme as both a regeneration-inducing therapeutic and a structure-directing agent in a supramolecular polymer that forms shear-thinning nanofiber hydrogels. Subcutaneous injection of the supramolecular hydrogel in the back of mice wounded with a critical-sized defect in the ear led to transient upregulation of hypoxia inducible factor-1α and regeneration of ear tissue in a manner reminiscent of epimorphic regeneration. This drug-induced regeneration strategy utilizes a simple and translatable supramolecular design, eliminates the need for delivery of biologics ( e. g., growth factors, cells), and avoids implantation of a foreign material directly in a tissue defect.

Drug delivery and epimorphic salamander-type mouse regeneration: A full parts and labor plan.

The capacity to regenerate entire body parts, tissues, and organs had generally been thought to be lost in evolution with very few exceptions (e.g. the liver) surviving in mammals. The discovery of the MRL mouse and the elucidation of the underlying molecular pathway centering around hypoxia inducible factor, HIF-1α, has allowed a drug and materials approach to regeneration in mice and hopefully humans. The HIF-1α pathway is ancient and permitted the transition from unicellular to multicellular organisms. Furthermore, HIF-1α and its regulation by PHDs, important oxygen sensors in the cell, provides a perfect drug target. We review the historical background of regeneration biology, the discovery of the MRL mouse, and its underlying biology, and novel approaches to drugs, targets, and delivery systems (see Fig. 1).

Oxygen, Metabolism, and Regeneration: Lessons from Mice.

The discovery that the Murphy Roths Large (MRL) mouse strain is a fully competent, epimorphic tissue regenerator, proved that the machinery of regeneration was preserved through evolution from hydra, to salamanders, to mammals. Such concepts have allowed translation of the biology of amphibians, and their ability to regenerate, to a mammalian context. We identified the ancient hypoxia-inducible factor (HIF)-1α pathway, operating through prolyl hydroxylase domain proteins (PHDs), as a central player in mouse regeneration. Thus, the possibility of targeting PHDs or other HIF-1α modifiers to effectively recreate the amphibian regenerative state has emerged. We posit that these regenerative pathways are critical in mammals. Moreover, the current approved use of PHD inhibitors in the clinic should allow fast-track translation from mouse studies to drug-based regenerative therapy in humans.

From Immunity and Vaccines to Mammalian Regeneration.

Our current understanding of major histocompatibility complex (MHC)-mediated antigen presentation in self and nonself immune recognition was derived from immunological studies of autoimmunity and virus-host interactions, respectively. The trimolecular complex of the MHC molecule, antigen, and T-cell receptor accounts for the phenomena of immunodominance and MHC degeneracy in both types of responses and constrains vaccine development. Out of such considerations, we developed a simple peptide vaccine construct that obviates immunodominance, resulting in a broadly protective T-cell response in the absence of antibody. In the course of autoimmunity studies, we identified the MRL mouse strain as a mammalian model of amphibian-like regeneration. A significant level of DNA damage in the cells from this mouse pointed to the role of the cell cycle checkpoint gene CDKN1a, or p21(cip1/waf1). The MRL mouse has highly reduced levels of this molecule, and a genetic knockout of this single gene in otherwise nonregenerating strains led to an MRL-type regenerative response, indicating that the ability to regenerate has not been lost during evolution.

Drug-induced regeneration in adult mice.

Whereas amphibians regenerate lost appendages spontaneously, mammals generally form scars over the injury site through the process of wound repair. The MRL mouse strain is an exception among mammals because it shows a spontaneous regenerative healing trait and so can be used to investigate proregenerative interventions in mammals. We report that hypoxia-inducible factor 1α (HIF-1α) is a central molecule in the process of regeneration in adult MRL mice. The degradation of HIF-1α protein, which occurs under normoxic conditions, is mediated by prolyl hydroxylases (PHDs). We used the drug 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), a PHD inhibitor, to stabilize constitutive expression of HIF-1α protein. A locally injectable hydrogel containing 1,4-DPCA was designed to achieve controlled delivery of the drug over 4 to 10 days. Subcutaneous injection of the 1,4-DPCA/hydrogel into Swiss Webster mice that do not show a regenerative phenotype increased stable expression of HIF-1α protein over 5 days, providing a functional measure of drug release in vivo. Multiple peripheral subcutaneous injections of the 1,4-DPCA/hydrogel over a 10-day period led to regenerative wound healing in Swiss Webster mice after ear hole punch injury. Increased expression of the HIF-1α protein may provide a starting point for future studies on regeneration in mammals.

Tendon regeneration and scar formation: The concept of scarless healing.

Tendon healing is characterized by the formation of fibrovascular scar tissue, as tendon has very little intrinsic regenerative capacity. This creates a substantial clinical challenge in the setting of large, chronic tears seen clinically. Interest in regenerative healing seen in amphibians and certain strains of mice has arisen in response to the biological behavior of tendon tissue. Bone is also a model of tissue regeneration as healing bone will achieve the mechanical and histologic characteristics of the original tissue. The ultimate goal of the study of genes and mechanisms that contribute to true tissue regeneration is to ultimately attempt to manipulate the expression of those genes and activate these mechanisms in the setting of tendon injury and repair. Clearly, further research is needed to bring this to the forefront, however, study of scarless healing has potential to have meaningful application to tendon healing.

Inflammation and Its Correlates in Regenerative Wound Healing: An Alternate Perspective.

Objective: The wound healing response may be viewed as partially overlapping sets of two physiological processes, regeneration and wound repair with the former overrepresented in some lower species such as newts and the latter more typical of mammals. A robust and quantitative model of regenerative healing has been described in Murphy Roths Large (MRL) mice in which through-and-through ear hole wounds in the ear pinna leads to scarless healing and replacement of all tissue through blastema formation and including cartilage. Since these mice are naturally autoimmune and display many aspects of an enhanced inflammatory response, we chose to examine the inflammatory status during regenerative ear hole closure and observed that inflammation has a clear positive effect on regenerative healing. Approach: The inflammatory gene expression patterns (Illumina microarrays) of early healing ear tissue from regenerative MRL and nonregenerative C57BL/6 (B6) strains are presented along with a survey of innate inflammatory cells found in this tissue type pre and postinjury. The role of inflammation on healing is tested using a COX-2 inhibitor. Innovation and Conclusion: We conclude that (1) enhanced inflammation is consistent with, and probably necessary, for a full regenerative response and (2) the inflammatory gene expression and cell distribution patterns suggest a novel mast cell population with markers found in both immature and mature mast cells that may be a key component of regeneration.

Hydrogels Formed by Oxo-ester Mediated Native Chemical Ligation.

Oxo-ester mediated native chemical ligation (OMNCL) is a variation of the more general native chemical ligation (NCL) reaction that is widely employed for chemoselective ligation of peptide fragments. While OMNCL has been used for a variety of peptide ligations and for biomolecular modification of surfaces, it is typically practiced under harsh conditions that are unsuitable for use in a biological context. In this report we describe the use of OMNCL for polymer hydrogel formation, in-vitro cell encapsulation, and in-vivo implantation. Multivalent polymer precursors containing N-hydroxysuccinimide (NHS) activated oxo-esters and N-cysteine (N-Cys) endgroups were chemically synthesized from branched poly(ethylene glycol) (PEG). Hydrogels formed rapidly at physiologic pH upon mixing of aqueous solutions of NHS and N-Cys functionalized PEGs. Quantitative 1H NMR experiments showed that the reaction proceeds through an OMNCL pathway involving thiol capture to form a thioester intermediate, followed by an S-to-N acyl rearrangement to yield an amide cross-link. pH and temperature were found to influence gelation rate, allowing tailoring of gelation times from a few seconds to a few minutes. OMNCL hydrogels initially swelled before contracting to reach an equilibrium increase in relative wet weight of 0%. This unique behavior impacted the gel stiffness and was attributed to latent formation of disulfide cross-links between network-bound Cys residues. OMNCL hydrogels were adhesive to hydrated tissue, generating a lap shear adhesion strength of 46 kPa. Cells encapsulated in OMNCL hydrogels maintained high viability, and in-situ formation of OMNCL hydrogel by subcutaneous injection in mice generated a minimal acute inflammatory response. OMNCL represents a promising strategy for chemical cross-linking of hydrogels in a biological context and is an attractive candidate for in-vivo applications such as wound healing, tissue repair, drug delivery, and tissue engineering.

Glioma grade is associated with the accumulation and activity of cells bearing M2 monocyte markers.

PURPOSE: This study is directed at identifying the cell source(s) of immunomodulatory cytokines in high-grade gliomas and establishing whether the analysis of associated markers has implications for tumor grading. EXPERIMENTAL DESIGN: Glioma specimens classified as WHO grade II-IV by histopathology were assessed by gene expression analysis and immunohistochemistry to identify the cells producing interleukin (IL)-10, which was confirmed by flow cytometry and factor secretion in culture. Finally, principal component analysis (PCA) and mixture discriminant analysis (MDA) were used to investigate associations between expressed genes and glioma grade. RESULTS: The principle source of glioma-associated IL-10 is a cell type that bears phenotype markers consistent with M2 monocytes but does not express all M2-associated genes. Measures of expression of the M2 cell markers CD14, CD68, CD163, and CD204, which are elevated in high-grade gliomas, and the neutrophil/myeloid-derived suppressor cell (MDSC) subset marker CD15, which is reduced, provide the best index of glioma grade. CONCLUSIONS: Grade II and IV astrocytomas can be clearly differentiated on the basis of the expression of certain M2 markers in tumor tissues, whereas grade III astrocytomas exhibit a range of expression between the lower and higher grade specimens. The content of CD163(+) cells distinguishes grade III astrocytoma subsets with different prognosis.

Keratin gene expression profiles after digit amputation in C57BL/6 vs. regenerative MRL mice imply an early regenerative keratinocyte activated-like state.

Mouse strains C57BL/6 (B6) and MRL were studied by whole mouse genome chip microarray analyses of RNA isolated from amputation sites at different times pre- and postamputation at the midsecond phalange of the middle digit. Many keratin genes were highly differentially expressed. All keratin genes were placed into three temporal response classes determined by injury/preinjury ratios. One class, containing only Krt6 and Krt16, were uniquely expressed relative to the other two classes and exhibited different temporal responses in MRL vs. B6. Immunohistochemical staining for Krt6 and Krt16 in tissue sections, including normal digit, flank skin, and small intestine, and from normal and injured ear pinna tissue exhibited staining differences in B6 (low) and MRL (high) that were consistent with the microarray results. Krt10 staining showed no injury-induced differences, consistent with microarray expression. We analyzed Krt6 and Krt16 gene association networks and observed in uninjured tissue several genes with higher expression levels in MRL, but not B6, that were associated with the keratinocyte activated state: Krt6, Krt16, S100a8, S100a9, and Il1b; these data suggest that keratinocytes in the MRL strain, but not in B6, are in an activated state prior to wounding. These expression levels decreased in MRL at all times postwounding but rose in the B6, peaking at day 3. Other keratins significantly expressed in the normal basal keratinocyte state showed no significant strain differences. These data suggest that normal MRL skin is in a keratinocyte activated state, which may provide it with superior responses to wounding.

Cell cycle regulation and regeneration.

Regeneration of ear punch holes in the MRL mouse and amputated limbs of the axolotl show a number of similarities. A large proportion of the fibroblasts of the uninjured MRL mouse ear are arrested in G2 of the cell cycle, and enter nerve-dependent mitosis after injury to form a ring-shaped blastema that regenerates the ear tissue. Multiple cell types contribute to the establishment of the regeneration blastema of the urodele limb by dedifferentiation, and there is substantial reason to believe that the cells of this early blastema are also arrested in G2, and enter mitosis under the influence of nerve-dependent factors supplied by the apical epidermal cap. Molecular analysis reveals other parallels, such as; (1) the upregulation of Evi5, a centrosomal protein that prevents mitosis by stabilizing Emi1, a protein that inhibits the degradation of cyclins by the anaphase promoting complex and (2) the expression of sodium channels by the epidermis. A central feature in the entry into the cell cycle by MRL ear fibroblasts is a natural downregulation of p21, and knockout of p21 in wild-type mice confers regenerative capacity on non-regenerating ear tissue. Whether the same is true for entry into the cell cycle in regenerating urodele limbs is presently unknown.

Heritability of articular cartilage regeneration and its association with ear wound healing in mice.

OBJECTIVE: Emerging evidence suggests that genetic components contribute significantly to cartilage degeneration in osteoarthritis pathophysiology, but little information is available on the genetics of cartilage regeneration. Therefore, this study was undertaken to investigate cartilage regeneration in genetic murine models using common inbred strains and a set of recombinant inbred (RI) lines generated from LG/J (healer of ear wounds) and SM/J (nonhealer) inbred mouse strains. METHODS: An acute full-thickness cartilage injury was introduced in the trochlear groove of 8-week-old mice (n=265) through microsurgery. Mouse knee joints were sagittally sectioned and stained with toluidine blue to evaluate regeneration. For the ear wound phenotype, a bilateral 2-mm through-and-through puncture was created in 6-week-old mice (n=229), and healing outcomes were measured after 30 days. Broad-sense heritability and genetic correlations were calculated for both phenotypes. RESULTS: Time-course analysis of the RI mouse lines showed no significant regeneration until 16 weeks after surgery; at that time, the strains could be segregated into 3 categories: good, intermediate, and poor healers. Analysis of heritability (H2) showed that both cartilage regeneration (H2=26%; P=0.006) and ear wound closure (H2=53%; P<0.00001) were significantly heritable. The genetic correlations between the two healing phenotypes for common inbred mouse strains (r=0.92) and RI mouse lines (r=0.86) were found to be extremely high. CONCLUSION: Our findings indicate that articular cartilage regeneration in mice is heritable, the differences between the mouse lines are due to genetic differences, and a strong genetic correlation between the two phenotypes exists, indicating that they plausibly share a common genetic basis. We therefore surmise that LG/J by SM/J intercross mice can be used to dissect the genetic basis of variation in cartilage regeneration.

The role of p21 in regulating mammalian regeneration.

The MRL (Murphy Roths Large) mouse has provided a unique model of adult mammalian regeneration as multiple tissues show this important phenotype. Furthermore, the healing employs a blastema-like structure similar to that seen in amphibian regenerating tissue. Cells from the MRL mouse display DNA damage, cell cycle G2/M arrest, and a reduced level of p21CIP1/WAF. A functional role for p21 was confirmed when tissue injury in an adult p21-/- mouse showed a healing phenotype that matched the MRL mouse, with the replacement of tissues, including cartilage, and with hair follicle formation and a lack of scarring. Since the major canonical function of p21 is part of the p53/p21 axis, we explored the consequences of p53 deletion. A regenerative response was not seen in a p53-/- mouse and the elimination of p53 from the MRL background had no negative effect on the regeneration of the MRL.p53-/- mouse. An exploration of other knockout mice to identify p21-dependent, p53-independent regulatory pathways involved in the regenerative response revealed another significant finding showing that elimination of transforming growth factor-ß1 displayed a healing response as well. These results are discussed in terms of their effect on senescence and differentiation.