Intestinal ischemia and necrosis affect multiple patient populations of varying ages and comorbidities. Acute mesenteric ischemia (AMI) is prevalent in the elderly population and those who undergo cardiac bypass surgery (1). AMI affects approximately 5,000 patients annually, with many requiring open or endovascular surgical intervention to lyse the clot and salvage the ischemic intestine. The mortality rate for AMI can be as high as 40% for those who progress to surgery (2). Necrotizing enterocolitis and volvulus are two forms of intestinal ischemia that can affect the neonatal population (3). Necrotizing enterocolitis, which ultimately manifests as intestinal ischemia and necrosis, affects the very low birth weight premature population. The mortality for the most severe cases of necrotizing enterocolitis can be quite high (4). Midgut volvulus associated with malrotation occurs much less frequently, but carries a high mortality when a majority of the bowel is involved (5). In any case, if injury is unrecognized or left untreated, patients can quickly decompensate and progress to shock, multisystem organ failure, and death. If patients survive these ischemic episodes, they are often faced with prolonged hospitalization and long-term parenteral nutrition needs (6).
Noteworthy advancements in the medical treatment of intestinal ischemia within the last decade have been sparse, and therefore stromal cell therapy offers a novel therapeutic option for the treatment of this disease (6). Bone marrow-derived mesenchymal stromal cells (BMSCs), in particular, have shown the capacity to promote survival and attenuate intestinal ischemic injury (3). These advantages are achieved, in part, through enhanced restitution and improved integrity of the intestinal mucosa, reduced translocation of bacteria from the lumen into the circulation, and a decreased inflammatory response (7, 8).
Despite their promising potential, BMSC use may be limited secondary to lower proliferative capacity and painful isolation procedures (9, 10). Adipose-derived mesenchymal stromal cells (ASCs), however, show greater proliferative potential than BMSCs and other mesenchymal stromal cell lines, and their ease of accessibility and limitless supply via liposuction of subcutaneous adipose tissue make them an ideal candidate for widespread therapeutic use (11–13). ASCs have also been suggested as an alternative stem cell source for the treatment of intestinal ischemia in a rat model of intestinal ischemia and reperfusion (I/R) (14). In this study, rat-derived ASCs decreased inflammation and preserved intestinal histological architecture, but the cellular effects on survival and mesenteric perfusion were not defined.
Adipose stromal cell transplants have also been shown to improve recovery of damaged tissue in several other models, including critical limb ischemia (15), acute kidney injury (16), cardiac ischemia (17), and stroke (18), but human ASCs (hASCs) have not yet been tested in preclinical models of intestinal ischemia. Therefore, the aim of the current study was to determine the efficacy of human ASC therapy in a murine model of intestinal I/R injury. We hypothesized that: hASCs would increase 7-day survival and mesenteric perfusion compared with differentiated cellular controls following intestinal I/R; improved outcomes with hASC therapy would be associated with preserved intestinal histology and tight junctional architecture following injury; and hASAC therapy following intestinal I/R would limit intestinal and systemic inflammation.
MATERIALS AND METHODS
Human ASCs were harvested from human subjects via liposuction and purified as previously described (19). Briefly, human subcutaneous adipose tissue samples obtained from liposuction procedures were digested in collagenase type I (Worthington Biochemical, Lakewood, NJ) under agitation for 2 h at 37°C. Samples were then centrifuged at 300g for 8 min to separate the stromal cell fraction (pellet) from adipocytes. The pellet was resuspended in DMEM/F12 containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) filtered through 250-μm Nitex filters (Sefar America Inc, Kansas City, MO) and centrifuged at 300g for an additional 8 min. The cell pellet was treated with red cell lysis buffer and was resuspended in EBM-2 with 5% FBS or EGM2-MV (Lonza, Allendale, NJ). Adipose stromal cells were CD34+, CD31−, and CD144− by flow cytometry, but also expressed several mesenchymal cell lineage markers including CD10, CD13, and CD90 (19). Cells were cultured on polystyrene flasks in Endothelial Growth Medium 2 MV with 5% FBS at 37°C in 5% CO2 in air. Cells were used between passages 4 and 7.
Human nTERT keratinocytes were graciously donated by Dr. Jeffery Travers at the Indiana University School of Medicine. Cells were originally purchased through ATCC (Manassas, VA) and were cultured in Epilife medium with keratinocyte growth factor (Life Technologies, Grand Island, NY). Cells were used between passages 24 and 35.
Once ready for experimentation, cell disassociation was achieved using TrypLE Express (Life Technologies). Cells were then pelleted at 400g for 5 min and resuspended in fresh media. Cells were then counted with the aid of an automated fluorescent cell counter (Luna-FL Automated Cell Counter, Logos Biosystems, Annandale, VA). Two million hASCs or keratinocytes were then resuspended in 250 μL of phosphate-buffered saline (PBS) vehicle for infusion as determined by a previous stromal cell dose response curve (3).
Murine intestinal I/R model
The experimental protocol and animal use were approved by the Indiana University Institutional Animal Care and Use Committee. Adult male C57Bl6J mice (8–12 wk, 20–30 g; Jackson Labs, Bar Harbor, ME) were allowed 48 h for acclimation to the new environment before intervention. They had access to normal chow and were kept in 12-h light-dark cycle housing. The animals were anesthetized at 3% isoflurane and maintained at 1.5% isoflurane for the duration of the procedure. Animals were placed on a heating pad to maintain body temperature during the procedure. The abdomen was prepped with hair removal lotion, followed by 70% ethanol and betadine. One milliliter of 0.9% normal saline was injected subcutaneously, and a midline laparotomy was performed.
The intestines were eviscerated, and the root of the superior mesenteric artery (SMA) was located. Temporary occlusion of the mesenteric root was established for 60 min using a noncrushing microvascular clamp. During ischemia, the abdomen was temporarily closed using silk suture to prevent evaporative heat and water loss. Following ischemia, the abdomen was reopened, the clamp was removed, and the intestines were allowed to recover. The abdominal fascia and skin were then closed in two layers using silk sutures. Before complete closure of the facial defect, 2 million hASCs, 2 million keratinocytes, or 250 μL of PBS vehicle were administered directly into the peritoneal cavity. Triple antibiotic ointment was applied to the abdominal incision site following complete closure, and analgesia (1 mg/kg buprenorphine and 5 mg/kg caprofen) was injected subcutaneously. Animals were recovered from anesthesia on the heating pad, placed back in their cage, and returned to animal housing.
Animals assigned to the survival protocol (n = 10 I/R + hASC, 10 I/R + keratinocytes, 10 I/R + PBS vehicle) were monitored twice daily over 7 days after the surgery for death, pain, and incisional complications. Endpoints of analysis included animal death or when Laboratory Animal Resource Center veterinarians felt that animals were suffering and needed to be euthanized. Survival curves were then created based on these endpoints.
Laser Doppler imaging analysis
Laser Doppler imaging (LDI) (Moor Instruments, Wilmington, DE) was used to assess blood perfusion of the intestines. At each designated time point (baseline, initiation of ischemia, initiation of reperfusion, 12 or 24 h of reperfusion), three LDI readings were taken for each animal, and an average perfusion value was calculated based on the flux mean of the three images. The flux mean value was a unit-less numerical value, with larger numbers representing greater perfusion, and smaller numbers representing lower perfusion or ischemia. The readings for each animal were expressed as a percentage of their baseline perfusion, with baseline representing 100% perfusion.
Animals assigned to the 12-h (N = 6/group) and 24-h (N = 7/group) reperfusion group were reanesthetized at 12 or 24 h, respectively. The incision site was reopened, and the intestines were eviscerated. LDI was then used to assess final perfusion parameters. Animals were then euthanized by isoflurane overdose and cervical dislocation. Animals that died before analysis were arbitrarily assigned a perfusion value of zero.
After 12 and 24 h of reperfusion, animals were euthanized and intestinal segments harvested. Segments were placed into 4% paraformaldehyde and subsequently dehydrated in 70% ethanol. Segments were then paraffin embedded and cut using a microtome. Tissue segments were placed on slides and were stained with hematoxylin and eosin. Histologic scoring of the depth of tissue injury was performed in blinded fashion by two of the authors as we have previously described (20): 0, no damage; 1, subepithelial space at the villous tip; 2, loss of mucosal lining of the villous tip; 3, loss of less than half of the villous structure; 4, loss of more than half of the villous structure; 5, transmural necrosis (N = 6–7 intestinal segments/group).
Paraffin-embedded small intestinal blocks (N = 6–7/group) were cut in 10 μm segments. Slides were deparaffinized with xylene and rehydrated in graded alcohols. Heat-induced epitope retrieval was then conducted in a standard pressure cooker and slides were placed in 10 mM citrate buffer (pH 6.0) for 20 min and allowed to cool. Slides were then blocked with normal Goat Serum (Biogenex, Fremont, CA) diluted in PBS with 1% bovine serum albumin (Santa Cruz Biotechnology, Dallas, TX) and 0.1% Tween 20 (Sigma, St. Louis, MO) for 1 h.
Presence of tight junctions in the intestinal tissues was then assessed by incubating slides with a 1:100 dilution of claudin-1 primary antibody (Novus Biologicals, NBP1-67515) overnight at 4°C. Following washing, Alexafluor 488 goat-anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA) was applied and incubated for 2 h at room temperature in a humidity chamber. Slides were then washed again and DAPI (4′,6-diamidino-2-phenylindole, Cell Signaling Technology) was applied at 1 μg/mL and allowed to incubate at room temperature for 5 min. Slides were then rinsed in PBS, mounted, coverslipped, and assessed using a fluorescent microscope. Staining was repeated on additional slides to ensure reproducible results.
Intestinal, liver, and lung tissue segments (N = 6–7/group) were thawed and homogenized in RIPA (radioimmunoprecipitation assay) buffer (Sigma) with protease and phosphatase cocktail inhibitors (1:100 dilution; Sigma). Homogenates were centrifuged at 12,000 rpm to pellet extraneous tissue, and supernatants were transferred to fresh eppendorff tubes for storage at −80°C. Total protein concentration was then quantified by Bradford Assay using a spectrophotometer (Versamax Microplate Reader, Molecular Devices). Tissue vascular endothelial growth factor (VEGF), granulocyte colony stimulating factor (GCSF), monokine induced by interferon gamma (MIG), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) were quantified with a Bioplex 200 multiplex beaded assay system (Bio-Rad, Hercules, Calif) using a customized multiplex kit for the designated chemokines (Millipore, Billerica, Mass). Assays were performed at 1:25 dilution according to the manufacturer's instructions.
Survival data were compared using the Mantel-Cox log-rank test and the Gehan-Breslow-Wilcoxon test. Reperfusion data were deemed to be normally distributed based on histogram data inspection, and were compared with two-way ANOVA and t test, when appropriate. Data are presented as percent change from baseline perfusion (mean% ± SEM). Histology and multiplex cytokine data were not normally distributed, and as such were compared using the Mann-Whitney U test. Data are presented as the median with 25% to 75% interquartile range. A P < 0.05 was considered statistically significant.
hASCs improved survival outcomes after intestinal I/R injury
Following intestinal I/R, 7-day survival was 40% in the vehicle group and 20% in those treated with keratinocytes. Seven-day survival significantly increased to 80% with the postischemic application of hASCs (Fig. 1; P < 0.05). These data identify hASCs as providing significant survival advantages to mice after intestinal I/R injury.
Postischemic mesenteric perfusion is improved with the use of hASC therapy
Administration of hASCs following intestinal I/R improved postischemic mesenteric perfusion at both 12 and 24 h of reperfusion compared with mice administered only vehicle (12-h reperfusion: PBS vehicle: 21.67 ± 10.22% vs. hASC: 71.00 ± 11.76%, P < 0.05; 24-h reperfusion: PBS vehicle: 25.57 ± 6.07% vs. hASC: 61.14 ± 11.89%, P < 0.05) (Fig. 2). Improved mesenteric perfusion was also appreciated at 24 h of reperfusion for the hASC group compared with the keratinocyte group (ASC: 61.14 ± 11.89% vs. keratinocyte: 25.00 ± 11.38%, P < 0.05), but this was not significant at 12-h postischemia. These results suggest that improved survival outcomes with hASC therapy following intestinal I/R may potentially be attributed to improved mesenteric perfusion following injury.
hASC therapy following intestinal I/R preserves intestinal tissue architecture
Intestinal I/R resulted in significant sloughing of the intestinal mucosa at both 12 and 24 h following reperfusion in vehicle-treated groups. This was seen as destruction of the epithelial layer in the crypt-villous architecture. Human ASC therapy following I/R abated this destruction and significantly decreased the median histology injury score (12-h reperfusion: PBS vehicle: 3.5 (25%–75%: 1.0–4.3) vs. hASC: 0 (25%–75%: 0.0–1.8), P < 0.05; 24-h reperfusion: PBS vehicle: 4.0 (25%–75%: 2.0–5.0) vs. hASC: 0 (25%–75%: 0.0–1.0), P < 0.05) (Fig. 3). Histology scores following keratinocyte therapy were similar to vehicle and were significantly worse than hASC groups. These results suggest that hASCs have a beneficial impact to the protection of intestinal mucosal integrity following I/R.
hASC therapy preserves intestinal tight junction architecture following injury
Animals exposed to hASCs following intestinal I/R injury demonstrated preservation of claudin-1 tight junctional proteins in the expected boarder zones between intestinal epithelial cells at 12 and 24 h of reperfusion. Cell junctions stained brightly with claudin-1 and the cell borders were crisply and clearly stained. Epithelial cells did not stain as strongly or as uniformly in PBS-treated groups. Keratinocyte-treated groups had decreased staining at 12 h, and evidence of hazy boarders between cells at 24 h, thereby suggesting disrupted gap junctions in these treated groups (Fig. 4).
hASC therapy affects intestinal, hepatic, and pulmonary inflammatory tissue cytokine production
Intestinal tissue levels of GCSF were significantly decreased at 24 h with the use of hASC therapy compared with PBS. Intestinal tissue levels of VEGF, MIG, IL-6, and IL-1β were not different between groups in intestinal tissues. Liver levels of GCSF were also significantly decreased in hASC groups at 24 h of reperfusion compared with PBS or keratinocyte groups. Liver levels of IL-1β were significantly lower in hASC groups compared with keratinocytes at 12 h of reperfusion, but no difference was seen at 24 h (Table 1).
hASC therapy seemed to have the most significant impact on pulmonary inflammation following intestinal I/R injury. Levels of GCSF, MIG, IL-6, and IL-1β were all significantly decreased in hASC groups at 24 h of reperfusion compared with PBS or keratinocyte groups. Levels of pulmonary VEGF were also significantly elevated in hASC groups at 24 h of therapy compared with PBS control, but were not significantly different from keratinocyte groups (Table 1).
Intestinal ischemia originates from multiple etiologies and can affect diverse patient demographics. No definitive medical advancements have been made in the treatment of intestinal ischemia in the last decade, and therefore the development of novel treatment modalities is paramount. The ultimate therapeutic goal in patients with intestinal ischemia is to restore blood flow to ischemic tissues before the development of necrosis and bowel wall perforation. In this study, we observed that hASCs significantly increased survival and mesenteric perfusion while simultaneously preserving intestinal histological and tight junction architecture compared with keratinocytes or vehicle in a murine model of intestinal I/R injury. In addition, hASCs abated the systemic inflammatory response, as seen predominantly by a decrease in lung tissue inflammatory markers.
Although previous studies have demonstrated the therapeutic benefits of hASCs in attenuation of other animal models of I/R injury (21), this is the first study to demonstrate the efficacy of human ASCs in rescuing murine intestinal ischemia. Previous studies have suggested that ASCs provide their benefit at least, in part, by the release of paracrine factors (22, 23). There are likely multiple growth factors and immune modulators that play a role in end-organ protection, and current studies by our group are attempting to define which of these factors may be the most efficacious in acute organ protection following injury.
Decreased mortality with hASC therapy was likely related to improved mesenteric perfusion and mucosal integrity. Improved perfusion likely restored blood flow and tissue oxygen levels to physiologic levels, which then prevented intestinal mucosal injury, sloughing, and the impending bacterial translocation and sepsis that would have ensued due to cell junction degradation. This concept was seen in this study by preservation of histological architecture and the tight junction protein claudin-1 in those animals treated with hASCs. It is unclear though, how the hASCs promoted improved mesenteric perfusion, but it may be, in part, to the release of specific vasodilators such as nitric oxide or hydrogen sulfide (24, 25) from these cells. Future studies aim to determine the specific properties of the hASCs that promote improved vascular perfusion and survival.
GCSF and MIG are two chemokines that are associated with inflammation and leukocyte infiltration, whereas IL-1β and IL-6 are notable proinflammatory cytokines associated with the acute phase response (26–29). hASC-treated groups exhibited a reduced systemic inflammatory response as noted by a decrease in these pulmonary inflammatory markers assessed at 24 h of reperfusion. It is unclear though as to why levels of these markers were not significantly altered in intestinal and hepatic tissues over the same time periods. Preservation of tight junctions and intestinal integrity with hASC therapy may have ameliorated the systemic inflammatory response and likely contributed to improvements in animal survival.
Clinical considerations to stromal cell therapy
The clinical use of hASCs in patients with intestinal ischemia provides a novel treatment option for a disease process that has not had a sound medical advancement in many years. Initial ASC clinical trials in other organ systems have been quite promising. Transendocardial injection of ASCs in patients with no-option ischemic cardiomyopathy indicated that autologous transplantation of ASCs was not only safe, but also preserved ventricular function, myocardial perfusion, and functional capacity (30). Another study using autologous ASCs in patients with nonrevascularizable critical limb ischemia demonstrated that ASCs improved wound healing, decreased pain, and expedited recovery time (31). Trials in patients with Crohn disease and complex perianal fistulas showed reduced fistula size and improved recovery when patients were administered ASCs (32).
Multiple animal studies and now several clinical trials have demonstrated extremely promising results with the use of ASC therapy. Challenges that face initiating clinical trials in human subjects with intestinal ischemia include identifying the most appropriate patients for whom to offer therapy. Many previous initial clinical trials have used patients in whom surgical revascularization was not an option. In the case of intestinal ischemia, patients who undergo surgical resection of necrotic bowel, but who have marginally ischemic boarder zones of intestine, may be the best candidates for initial therapeutic trials. With the aid of second look laparotomy, surgeons could apply the cells on the day of intestinal resection, temporarily close the abdomen, and return to the operating room in 24 to 48 h to assess the viability of the remaining intestines.
An additional challenge to therapy is identifying the optimal stromal cell source (bone marrow, adipose tissue, umbilical cord), as well as determining the donor type for therapy (allogenic versus autologous). Risks to allogenic therapy include potential immunogenic reactions to foreign cells, although this is less likely given the ability of mesenchymal stromal cells to downregulate T-cell proliferation (33). Finally, risks of malignancy are always considered with stromal cell therapy given the highly proliferative potential of these cells. The risks of malignancy in the future would need to be balanced with the risks of mortality and morbidity at the time of therapeutic use.
Limitations to this study
This study has several limitations that may affect the impact of the results. The first limitation is that human cells were used as a preclinical assessment in a mouse model of intestinal I/R injury. Although xenotransplantation may be less desirable, the use of stromal cells from animals is certainly feasible. Mesenchymal stromal cells have unique immunomodulatory properties that suppress T-lymphocyte proliferation and allow them to be used in different species (33). There have been over 25 different studies where human mesenchymal stromal cells have been placed into immunocompetent hosts of different species (3, 34). Interestingly, in the majority of these cases, the cells survived several weeks and engrafted into the host tissue (35).
A subsequent limitation to the study is that the SMA ligation model of intestinal I/R does not model clinical intestinal ischemia to its fullest. Although complete small bowel ischemia is possible secondary to SMA thrombus or embolus, the majority of intestinal ischemic episodes are due to segmental intestinal ischemia, such as may be seen with adhesive bowel obstructions or incarcerated hernias. Nonetheless, the SMA ligation model mimics the most severe form of intestinal ischemia, and therefore is likely the best animal model available to test the effectiveness of new therapies.
In addition, a limitation exists in the assessment of tissue cytokines. Despite normalizing for total protein concentration, a wide variation of levels was observed between group samples. Although the same relative area of intestine, liver, and lung was procured from each subject, it is likely that tissue levels of cytokines are not exactly equal throughout even small segments of tissue.
Adipose-derived stromal cells have shown promise as a potential novel treatment option for patients with intestinal ischemia. More specifically, we observed that human ASCs improve survival outcomes, improve mesenteric perfusion and intestinal histologic architecture, and decrease systemic inflammation. Clinical trials that use hASCs for the treatment of intestinal ischemia are certainly on the horizon, but a clear understanding of the mechanism of action must be addressed to ensure the safety and efficacy of therapy before widespread clinical use.
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