Modulation of the inflammatory response by increasing fetal wound size or interleukin-10 overexpression determines wound phenotype and scar formation.

Wound size impacts the threshold between scarless regeneration and reparative healing in the fetus with increased inflammation showed in fetal scar formation. We hypothesized that increased fetal wound size increases pro-inflammatory and fibrotic genes with resultant inflammation and fibroplasia and that transition to scar formation could be reversed by overexpression of interleukin-10 (IL-10). To test this hypothesis, 2-mm and 8-mm dermal wounds were created in mid-gestation fetal sheep. A subset of 8-mm wounds were injected with a lentiviral vector containing the IL-10 transgene (n = 4) or vehicle (n = 4). Wounds were harvested at 3 or 30 days for histology, immunohistochemistry, analysis of gene expression by microarray, and validation with real-time polymerase chain reaction. In contrast to the scarless 2-mm wounds, 8-mm wounds showed scar formation with a differential gene expression profile, increased inflammatory cytokines, decreased CD45+ cells, and subsequent inflammation. Lentiviral-mediated overexpression of the IL-10 gene resulted in conversion to a regenerative phenotype with decreased inflammatory cytokines and regeneration of dermal architecture. In conclusion, increased fetal wounds size leads to a unique gene expression profile that promotes inflammation and leads to scar formation and furthermore, these results show the significance of attenuated inflammation and IL-10 in the transition from fibroplasia to fetal regenerative healing.

Mammalian fetal cardiac regeneration after myocardial infarction is associated with differential gene expression compared with the adult.

BACKGROUND: In adults, myocardial infarction (MI) results in a brisk inflammatory response, myocardium loss, and scar formation. We have recently reported the first mammalian large-animal model of cardiac regeneration after MI in fetal sheep. We hypothesize that the ability of the fetus to regenerate functional myocardium after MI is owing to differential gene expression regulating the response to MI in the fetus compared with the adult. METHODS: Myocardial infarction was created in adult (n=4) or early gestation fetal (n=4) sheep. Tissue was harvested after 3 or 30 days, and RNA was extracted for microarray, followed by principal component analysis and global gene expression analysis for the following gene ontology terms: response to wounding, inflammatory response, extracellular matrix, cell cycle, cell migration, cell proliferation, and apoptosis. RESULTS: Principal component analysis demonstrated that the global gene expression pattern in adult infarcts was distinctly different from the uninfarcted region at 3 days and remained different at 30 days after MI. In contrast, gene expression in the fetal infarct was different from the uninfarcted region at 3 days, but by 30 days it returned to a baseline expression pattern similar to the uninfarcted region. Three days after MI there was an increase in the expression of genes related to all gene ontology terms in fetal and adult infarcts, but this increase was much more pronounced in adults. By 30 days, the fetal gene expression returned to baseline, whereas in the adult it remained significantly elevated. CONCLUSIONS: These data demonstrate that the global gene expression pattern is dramatically different in the fetal regenerative response to MI compared with the adult response and may partly be responsible for the regeneration.

The fate of untreated concomitant suprarenal aortic aneurysms after endovascular aneurysm repair of infrarenal aortic aneurysms.

OBJECTIVE: Many patients treated with endovascular aortic repair (EVAR) have a concomitant suprarenal abdominal aortic aneurysm (sAAA). The natural history of these sAAAs and whether they require intervention after EVAR is unknown. METHODS: We identified 470 patients from the M2S database (M2S Inc, West Lebanon, NH) as having an infrarenal AAA (iAAA) with a concomitant sAAA (diameter, 2.9-4.7 cm). The analysis included 217 patients with preoperative computed tomography angiography and follow-up imaging of ≥12 months. Patients who did not undergo EVAR (n = 65) served as a control. Patients with EVAR were subdivided into 90 with suprarenal fixation (SR) and 62 with infrarenal fixation (IR). Standard measurements from the M2S images were extracted, and growth rates were calculated for different abdominal aortic segments. RESULTS: The average follow-up was 33.0 ± 18.8 months. The average sAAA initial size and growth rate were 34.6 ± 3.0 and 0.6 ± 1.1 mm/y for SR, 34.0 ± 3.3 and 0.6 ± 1.3 mm/y for IR, and 36.6 ± 3.4 and 1.2 ± 1.5 mm/y for controls (SR vs IR, P > .05; SR or IR vs control, P < .05). After EVAR, two of 152 (1.3%) sAAAs grew to ≥ 50 mm, which was not statistically different from four of 65 (6.2%) in the control group (P = .07). At 48 months, the Kaplan-Meier freedom from sAAA growth to ≥ 50 mm was 99.3% for patients undergoing EVAR and 95.2% for controls (P = .061). Patients with starting sAAAs sized ≥ 40 mm had a higher growth rate (1.4 ± 2.1 mm/y) and frequency of growth to ≥50 mm (14.3%) than patients with starting sAAAs sized <40 mm (0.7 ± 1.2 mm/y and 1.5%; P < .05). CONCLUSIONS: Isolated treatment of iAAAs via EVAR with a concomitant sAAA is acceptable because endografts with or without SR do not affect sAAA growth rates. Routine EVAR follow-up is sufficient for sAAAs of <40 mm, and more intensive follow-up should be considered for sAAAs of 40 to 50 mm. For sAAAs approaching 50 mm, an endograft with IR should be considered in case sAAA repair is required in the future.

The management of endograft infections following endovascular thoracic and abdominal aneurysm repair.

OBJECTIVE: The management of infected aortic endografts is a challenging endeavor. Treatment of this problem has not been well defined as it is fairly uncommon. However, the incidence is increasing. This study examines the results of treatment at a single center for this morbid process. METHODS: A retrospective review was performed of patients treated for infected abdominal or thoracic endograft infection following previous abdominal or thoracic endovascular aneurysm repair. Data was reviewed for patient demographics, details of initial endograft implantation, presentation and timeline of subsequent infection, management of infected grafts, and outcomes during follow-up. RESULTS: Overall, 18 patients were treated for infected endografts (thoracic: six, abdominal:12). Three patients were treated between 2000 and 2006, corresponding to a 0.6% institutional incidence of endograft infection (3/473). There were no transfers for infected endografts from outside institutions. From 2006 to 2011, 15 patients underwent treatment. Six were institutional cases of infections (6/945, 0.6% infection rate), however, there was an increase in transfers (n = 9). Median time to presentation with infection from endograft implant was 90 days, with over one-half (61%) presenting within the first 3 months. Tissue and/or blood cultures were positive in 12/16 growing Escherichia coli (n = 1), group A streptococcus (n = 3), methicillin-resistant Staphylococcus aureus (n = 3), or polymicrobial infections (n = 7). The other four patients were culture negative with computed tomography evidence of gas surrounding the endograft and clinical sepsis. Ten patients (abdominal: eight, thoracic: two) were treated with endograft explantation. The remaining eight patients were considered too high-risk for explant or refused open surgery and were therefore managed conservatively without explant (abdominal: four, thoracic: four). At a mean follow-up of 24.7 months, aneurysm-related mortality was 38.9% (n = 7) and was higher for patients presenting with aortoenteric or aortobronchial fistulas (n = 6/10, 60%) (P = .04) and for thoracic stent infections (n = 5/6; 83%) (P = .03). The only survivor of a thoracic infection was managed surgically. Overall survival for patients with abdominal endografts (n = 12) was similar between the eight patients managed surgically (n = 6/8; 75%) and the four selected for medical management (n = 4/4; 100%) (P = .39). All survivors remain on long-term suppressive antibiotics. Two additional patients died of unrelated causes during follow-up. CONCLUSIONS: Endograft infection is a rare but increasing complication after abdominal or thoracic endovascular aneurysm repair, which carries significant associated morbidity and mortality. Most endograft infections occurred in proximity to other types of infection, suggesting that bacterial seeding of the endograft was the source. Aortoenteric and aortobronchial fistulas are common presentations, which portend a significantly worse prognosis. Thoracic endograft infections, which have the highest rate of fistulization, have the worst outcomes. Surgical excision continues to be standard of care but conservative management with intravenous antibiotics may be of benefit in certain patients with abdominal endograft infections.

Lentiviral-mediated over-expression of hyaluronan synthase-1 (HAS-1) decreases the cellular inflammatory response and results in regenerative wound repair.

Fetal wounds have been found to have increased levels of high-molecular-weight hyaluronan (HMW-HA) compared with those of adults. The primary enzyme responsible for producing HMW-HA is hyaluronic acid synthase-1 (HAS-1). We hypothesized that over-expression of HAS-1 in adult dermal wounds would decrease inflammation and promote regenerative healing. To test this hypothesis, the flanks of adult C57Bl/6 mice were treated with a lentiviral construct containing either HAS-1-GFP or GFP transgenes. After 48 h, a 4-mm excisional wound was made at the site of treatment. Wounds were harvested at days 3, 7, or 28 after wounding. Wound phenotype was assessed by histology to examine tissue architecture and immunohistochemistry for CD45. At 7 and 28 days, lenti-HAS-1-treated wounds demonstrated the restoration of the normal dermal elements and organized collagen fiber orientation. In contrast, the lenti-GFP-treated wounds lacked normal dermal architecture and demonstrated a disorganized collagen scar. At 3 and 7 days, wounds treated with lenti-HAS-1 exhibited a significant decrease in the number of inflammatory cells when compared with wounds treated with lenti-GFP. Thus, HAS-1 over-expression promotes dermal regeneration, in part by decreasing the inflammatory response and by recapitulation of fetal extracellular matrix HMW-HA content.

Impaired biomechanical properties of diabetic skin implications in pathogenesis of diabetic wound complications.

Diabetic skin is known to have deficient wound healing properties, but little is known of its intrinsic biomechanical properties. We hypothesize that diabetic skin possesses inferior biomechanical properties at baseline, rendering it more prone to injury. Skin from diabetic and nondiabetic mice and humans underwent biomechanical testing. Real-time PCR was performed for genes integral to collagen synthesis and degradation. MMP-2 and MMP-9, and TIMP-1 protein levels were assessed by ELISA and zymography. Collagen I and III content was assessed using Western blot analysis. At baseline, both murine and human diabetic skin was biomechanically inferior compared to nondiabetic skin, with decreased maximum stress and decreased modulus (P < 0.001 and < 0.05, respectively). Surprisingly, the expression of genes involved in collagen synthesis were significantly up-regulated, and genes involved in collagen degradation were significantly down-regulated in murine diabetic skin (P < 0.01). In addition, MMP-2 and MMP-9/TIMP-1 protein ratios were significantly lower in murine diabetic skin (P < 0.05). Collagen I levels and I:III ratios were lower in diabetic skin (P < 0.05). These findings suggest that the predisposition of diabetics to wounds may be the result of impaired tissue integrity at baseline, and are due, in part, to a defect in the regulation of collagen protein synthesis at the post-transcriptional level.

Inhibition of stromal cell-derived factor-1α further impairs diabetic wound healing.

OBJECTIVE: Impaired diabetic wound healing is associated with abnormal stromal cell-derived factor (SDF)-1α production, decreased angiogenesis, and chronic inflammation. Lentiviral-mediated overexpression of SDF-1α can correct the impairments in angiogenesis and healing in diabetic wounds. We hypothesized that SDF-1α is a critical component of the normal wound-healing response and that inhibition of SDF-1α would further delay the wound-healing process. METHODS: dB/Db diabetic mice and Db/+ nondiabetic mice were wounded with an 8-mm punch biopsy and the wounds treated with a lentiviral vector containing either the green fluorescent protein (GFP) or SDF-1α inhibitor transgene. The inhibitor transgene is a mutant form of SDF-1α that binds, but does not activate, the CXCR4 receptor. Computerized planimetry was used to measure wound size daily. Wounds were analyzed at 3 and 7 days by histology and for production of inflammatory markers using real-time polymerase chain reaction. The effect of the SDF-1α inhibitor on cellular migration was also assessed. RESULTS: Inhibition of SDF-1α resulted in a significant decrease in the rate of diabetic wound healing, (3.8 vs 6.5 cm(2)/day in GFP-treated wounds; P = .04), and also impaired the early phase of nondiabetic wound healing. SDF-1α inhibition resulted in fewer small-caliber vessels, less granulation tissue formation, and increased proinflammatory gene expression of interleukin-6 and macrophage inflammatory protein-2 in the diabetic wounds. CONCLUSIONS: The relative level of SDF-1α in the wound plays a key role in the wound-healing response. Alterations in the wound level of SDF-1α, as seen in diabetes or by SDF-1α inhibition, impair healing by decreasing cellular migration and angiogenesis, leading to increased production of inflammatory cytokines and inflammation. Inhibition of SDF-1α further impairs diabetic wound healing.

Fetal tendon wound size modulates wound gene expression and subsequent wound phenotype.

The fetal response to small tendon injury results in regenerative or scarless healing and is characterized by a markedly diminished cellular inflammatory response, lack of fibroplasia, and restoration of normal tissue architecture. We hypothesized that an increasing fetal tendon wound size would lead to increased wound inflammation and a change from regenerative to reparative healing and scar formation. We created small or large tendon wounds in early gestation fetal sheep and used histology to assess tissue architecture, immunohistochemistry to assess the cellular inflammatory response, ovine-specific gene microarrays, and real-time reverse transcription-polymerase chain reaction to measure the gene expression in response to injury. Small tendon wounds showed a regenerative healing phenotype with orderly deposition of collagen fibers while large tendon wounds showed disorderly collagen deposition consistent with scar formation. Small tendon wounds had few inflammatory cells at 7 and 28 days after injury, whereas the large wounds showed a significant inflammatory cell infiltrate at 7 days that resolved by 28 days. At 3 days, the differential expression of genes involved in the response to injury and inflammation were seen between large and small tendon wounds. By real-time polymerase chain reaction at 7 days, large tendon wounds also had significantly increased expression of interleukin-6, interleukin-8, transforming growth factor-ß1, and transforming growth factor-ß3, compared with the small wounds. Increasing the fetal tendon wound size results in increased proinflammatory gene expression, inflammatory cell infiltration, and a change from regenerative to reparative healing. This model allows the process of regenerative healing to be examined without the confounding variable of gestational age.

Regenerative healing following foetal myocardial infarction.

OBJECTIVES: The adult response to myocardial infarction results in inflammation, scar formation, left ventricular dilatation, and loss of regional and global function. Regenerative scarless healing has been demonstrated in foetal dermis and tendon and is associated with diminished inflammation. We hypothesised that following foetal myocardial infarction, there would be minimal inflammation, regenerative healing, and preservation of function. METHODS: Anteroapical myocardial infarction encompassing 20% of the left ventricle was created in adult or early gestation foetal sheep. Myocardial function was serially assessed using quantitative echocardiography. Infarct architecture was examined histologically for evidence of scar formation. Cellular inflammation, cellular proliferation, and apoptosis were assessed using immunohistochemistry. RESULTS: In the adult sheep 4 weeks following myocardial infarction, there was a significant decline in ejection fraction (EF) (41±7.4% to 26±7.4%, p<0.05), and the akinetic myocardial segment increased in size (6.9±0.8 cm to 7.9±1.1 cm, p<0.05). By contrast, there was no decline in the foetal EF (53±8.1% to 55±8.8%) and no akinetic foetal myocardial segment 4 weeks post-infarction. The foetal infarcts lacked an inflammatory cell infiltrate and healed with minimal fibrosis, compared with the adults. Foetal infarcts also demonstrated 5-bromo-2'-deoxyuridine (BrdU)+ proliferating cells, including cardiomyocytes, within the infarct. CONCLUSIONS: These data demonstrate that the foetal response to myocardial infarction is dramatically different from the adult and is characterised by minimal inflammation, lack of fibrosis, myocardial proliferation and restoration of cardiac function. Diminished inflammation is associated with foetal regenerative cardiac healing following injury. Understanding the mechanisms involved in foetal myocardial regeneration may lead to applications to alter the adult response following myocardial infarction.

Progenitor Cells for the Treatment of Acute Myocardial Infarction.

BACKGROUND: Left ventricular remodeling after myocardial infarction (MI) results in ventricular dilation, fibroplasia, and decline in cardiac function. There is significant morbidity and mortality associated with this process, often leading to heart failure despite medical management. New therapies aimed at altering ventricular remodeling after MI are needed, and cytotherapy utilizing progenitor cells is a current area of investigation. THE PROBLEM: Many variables need to be considered to maximize the therapeutic benefit of cytotherapy, including the cell type, the method of delivery, the timing, and patient selection. Investigation into progenitor cell biology will continue to identify novel treatment strategies that then must be tested clinically to demonstrate safety, feasibility, and ultimately therapeutic benefit. BASIC/CLINICAL SCIENCE ADVANCES: Although progenitor cells have the potential to affect ventricular remodeling through multiple mechanisms, their major effect is likely due to actions of secreted factors. Hepatocyte growth factor, stromal-derived growth factor-1 alpha, and stem cell factor affect the mobilization of progenitor cells, which makes it possible to develop treatment strategies based on recruitment of endogenous progenitor cells. CLINICAL CARE RELEVANCE: There have been a number of randomized controlled trials demonstrating modest improvements in cardiac function using progenitor cell therapies after MI. In addition, clinical trials have used granulocyte-colony-stimulating factor as a treatment aimed at increasing progenitor cell mobilization. CONCLUSION: Progenitor cell therapy after MI has been shown to be safe and feasible with some improvements in cardiac function and clinical outcomes. Further investigation is needed to better understand the biology of progenitor cells and the effects of progenitor cell-based therapies.