Novel Regenerative Peptide TP508 Mitigates Radiation-Induced Gastrointestinal Damage by Activating Stem Cells and Preserving Crypt Integrity

Carla Kantara; Stephanie M Moya; Courtney W Houchen; Shahid Umar; Robert L Ullrich; Pomila Singh; Darrell H Carney

Disclosures

Lab Invest. 2015;95(11):1222-1233. 

In This Article

Abstract and Introduction

Abstract

In recent years, increasing threats of radiation exposure and nuclear disasters have become a significant concern for the United States and countries worldwide. Exposure to high doses of radiation triggers a number of potentially lethal effects. Among the most severe is the gastrointestinal (GI) toxicity syndrome caused by the destruction of the intestinal barrier, resulting in bacterial translocation, systemic bacteremia, sepsis, and death. The lack of effective radioprotective agents capable of mitigating radiation-induced damage has prompted a search for novel countermeasures that can mitigate the effects of radiation post exposure, accelerate tissue repair in radiation-exposed individuals, and prevent mortality. We report that a single injection of regenerative peptide TP508 (rusalatide acetate, Chrysalin) 24 h after lethal radiation exposure (9 Gy, LD100/15) appears to significantly increase survival and delay mortality by mitigating radiation-induced intestinal and colonic toxicity. TP508 treatment post exposure prevents the disintegration of GI crypts, stimulates the expression of adherens junction protein E-cadherin, activates crypt cell proliferation, and decreases apoptosis. TP508 post-exposure treatment also upregulates the expression of DCLK1 and LGR5 markers of stem cells that have been shown to be responsible for maintaining and regenerating intestinal crypts. Thus, TP508 appears to mitigate the effects of GI toxicity by activating radioresistant stem cells and increasing the stemness potential of crypts to maintain and restore intestinal integrity. These results suggest that TP508 may be an effective emergency nuclear countermeasure that could be delivered within 24 h post exposure to increase survival and delay mortality, giving victims time to reach clinical sites for advanced medical treatment.

Introduction

The threat of a nuclear incident, with the potential to kill or injure thousands of people, has increased national and international recognition of the need for medicinal countermeasures that can prevent radiation-induced tissue damage and keep people alive, even if administered a day or more after nuclear exposure.[1,2] Exposure to high doses of total body radiation (≥9 Gy) triggers an acute gastrointestinal radiation toxicity syndrome (GI toxicity) that often results in death, regardless of intervention with advanced therapeutics or bone marrow transplants.[3,4] The high mortality associated with GI toxicity is believed to be caused by the radiation-induced damage to the intestinal and colonic mucosa leading to reduced fluid absorption, electrolyte imbalance, barrier function loss, bacterial translocation, systemic bacterial infection, sepsis, and organ failure.[5–8] This sequence in the GI system is initiated by radiation-induced damage to stem cells that must continually proliferate to maintain crypt integrity and regeneration.[9,10] Crypt cells in both the small intestine and colon are susceptible to radiation damage and serve as an indicator of potential survival following total body radiation.[11] Hence, it is crucial to develop novel therapeutic drugs capable of preventing damage to GI crypt stem cells in order to increase survival.

To date, only a few mitigating or radioprotective agents have been approved by the FDA.[12,13] Most of these are only effective in treating the hematopoietic syndrome triggered by low dose radiation, are unsuccessful in treating GI toxicity induced by high-dose radiation exposures, or are ineffective as a post-exposure treatment for the thousands of potential exposed individuals.[12,13]

TP508 (Chrysalin) is an investigational peptide drug that was developed for use in stimulating repair of dermal and musculoskeletal tissues.[14] TP508 is a 23-amino acid peptide representing amino acids 508–530 of human prothrombin that was identified as the high-affinity binding domain of thrombin responsible for interaction with a subset of thrombin receptors on the surface of fibroblasts thought to initiate tissue repair.[15,16] Specificity of TP508 has been demonstrated in both in vitro and in vivo experiments by altering the sequence and/or using scrambled peptides.[17–20] TP508 was shown to initiate tissue repair and regeneration by reversing endothelial dysfunction,[21] stimulating revascularization,[22–24] attenuating inflammation,[25] and reducing apoptosis.[26] In human clinical trials, TP508 was shown to significantly increase healing of diabetic foot ulcers[14,24,27] and distal radius fractures with no drug-related adverse events.[14,24] Animal studies also showed that TP508 treatment regenerated bone in critical-size defects where new bone formation would not occur without intervention.[28] Recently, this 23-amino acid regenerative peptide has been shown to target stem/progenitor cells isolated from tissues and stimulate their proliferation.[29] Thus, many of the tissue repair and regeneration effects of TP508 may be mediated by the activation of progenitor/stem cells within tissues.

It is well established that high-dose radiation exposure disrupts the normal homeostasis of crypts in the small intestine and colon.[30] Certain growth factors and cytokines have been reported to have protective effects against radiation-induced damage to the intestinal epithelium.[31] These factors are known to stimulate the proliferation of stem cells within the intestinal crypts.[32,33] Given that TP508 stimulates stem cell proliferation[29] and regeneration of tissues, we hypothesized that TP508 may protect intestinal crypts or accelerate their regeneration by the upregulation of stem/progenitor cells to mitigate lethal effects of radiation exposure.

In this study, we show that TP508 effectively protects the intestinal mucosa from radiation-induced damage by increasing crypt stem cell proliferation, rescuing the stemness potential of the crypt cells, and preventing crypt disintegration post radiation exposure by maintaining E-cadherin adherens junctions. These protective effects of TP508 are seen in intestinal crypts (Supplementary Figures 1 and 2 http://www.nature.com/labinvest/journal/v95/n11/suppinfo/labinvest2015103s1.html) and in colonic crypts (Figures 1, 2, 3 and 4) following 9 Gy (LD100/15) exposures. Importantly, mice treated with TP508 24 h post 9 Gy exposure show a significant delay in the onset of mortality and a significant increase in survival. Therefore, TP508 may be an effective post-exposure medicinal countermeasure for mitigating radiation-induced GI damage and mortality following a nuclear incident.

Figure 1.

Effects of TP508 on gastrointestinal colonic crypts integrity post-radiation exposure. (a) Representative images taken at × 10 and × 40 magnifications of intact colonic crypts harvested at 48 h, 5 days and 9 days post-RT from mice treated with either Saline or TP508, 24 h post radiation (0 or 9 Gy). (bi–ii) Representative H&E staining of colonic crypts sections harvested at 48 h, day 5 and 9 days post-RT, from mice treated with the indicated treatments. Inset illustrating H&E images from colonic crypts isolated 5 days post-RT is shown in the right-hand panel. White arrows depict change in crypt lengths. (b ii) Bar graphs showing the percent change in crypt lengths normalized to the control (0 Gy+Saline) group, isolated 48 h, 5 days and 9 days post-RT, respectively. Data=mean±s.e.m. from 6 mice/group/3 experiments. *P<0.05 vs 9 Gy+Saline values.

Figure 2.

TP508 increases the expression of adherens junction E-cadherin and decreases apoptosis in gastrointestinal crypts post radiation exposure. (ai) Representative immunofluorescent images of colonic crypt sections harvested 48 h, 5 days and 9 days post-RT and stained for E-cadherin. (aii) Bar graphs illustrating the % change in the number of E-cadherin positive cells per crypt normalized to the control group (0 Gy+Saline). (bi) Immunofluorescent staining of colonic crypts sections harvested at 48 h, 5 days and 9 days post-RT from mice treated with the indicated treatments for apoptotic marker activated-caspase-3. (bii) Bar graphs showing the percent change in the number of activated caspase-3 positive cells per crypt normalized to the control (0 Gy+Saline) group, isolated 48 h, 5 days and 9 days post-RT, respectively. Data=mean±s.e.m. from 6 mice/group/3 experiments. *P<0.05 vs 9 Gy+Saline values.

Figure 3.

TP508 stimulates the proliferation of gastrointestinal crypt cells post radiation exposure. (ai) Representative images of colonic crypts sections harvested 48 h, 5 days and 9 days post-RT from mice treated with the indicated treatments were stained for PCNA. (aii) Bar graphs showing the percent change in the number of PCNA positive cells per crypt normalized to the control (0 Gy+Saline) group, isolated 48 h, 5 days and 9 days post-RT, respectively. Data=mean±s.e.m. from 6 mice/group/3 experiments. *P<0.05 vs 9 Gy+Saline values. Ratio of control samples (0 Gy+Saline) were arbitrarily assigned 100% values; ratios of treated samples were expressed as a % of the control group. *P<0.05 vs control (9 Gy+Saline) values.

Figure 4.

TP508 increases the stemness and proliferative potential of intact colonic crypts post radiation exposure while decreasing apoptosis. (a) Western blot (WB) analysis demonstrating the expression of the indicated markers in Saline- vs TP508-treated groups at 48 h and 9 days post-RT. (bi, ii) Mean±s.e.m. of WB data from 4 mice/group/3 experiments, presented as % change in ratio of target protein/β-actin from samples collected 48 h (i) and 9 days (ii) post-RT. Ratio of control samples (0 Gy+Saline) were arbitrarily assigned 100% values; ratios of treated samples were expressed as a % of control. *P<0.05 vs control (9 Gy+Saline) values.

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