Hepatocyte autophagy is linked to C/EBP-homologous protein, Bcl2-interacting mediator of cell death, and BH3-interacting domain death agonist gene expression.

BACKGROUND: Endoplasmic reticulum (ER) stress and autophagy each play important roles in hepatocyte cell injury. We hypothesized that gene expression of C/EBP-homologous protein (CHOP) and the BH3 proteins Bcl2-interacting mediator of cell death (BIM) and BH3-interacting domain death agonist (BID) are involved in a complex interplay that regulates ER stress-induced autophagy and cell death. MATERIALS AND METHODS: Hepatocytes were cultured from lean Zucker rats. Confluent hepatocytes were incubated with single or combined small interfering RNA for CHOP, BIM, and/or BID for 24 h providing gene inhibition. Incubation with tunicamycin (TM) for another 24 h stimulated ER stress. Quantitative real-time polymerase chain reaction determined the expression levels of CHOP, BIM, and BID. Immunostaining with microtubule-associated protein 1 light chain 3 measured autophagy activity. Trypan blue exclusion determined the cell viability. RESULTS: TM treatment increased the messenger RNA levels of CHOP and BIM but decreased the messenger RNA levels of BID. TM increased autophagy and decreased cell viability. Individual inhibition of CHOP, BIM, or BID protected against autophagy and cell death. However, simultaneous treatment with any combination of CHOP, BIM, and BID small interfering RNAs reduced autophagy activity but increased cell death independent of ER stress induction. CONCLUSIONS: Autophagy in hepatocytes results from acute ER stress and involves interplay, at the gene expression level, of CHOP, BIM, and BID. Inhibition of any one of these individual genes during acute ER stress is protective against cell death. Conversely, inhibition of any two of the three genes results in increased nonautophagic cell death independent of ER stress induction. This study suggests interplay between CHOP, BIM, and BID expression that can be leveraged for protection against ER stress-related cell death. However, disruption of the CHOP/BH3 gene expression homeostasis is detrimental to cell survival independent of other cellular stress.

Dysregulation of collagen production in diabetes following recurrent skin injury: contribution to the development of a chronic wound.

Recurrent injury has been implicated in the development of chronic diabetic wounds. We have developed a chronic diabetic wound model based upon recurrent injury in diabetic mice. We hypothesized that dysregulation of collagen production at both the mRNA and microRNA levels contributes to the development of chronic diabetic wounds. To test this, both diabetic and nondiabetic mice were made to undergo recurrent injury. Real-time PCR for TGF-ß1, SMAD-3, Col1α1, Col3α1, microRNA-25, and microRNA-29a and Western blot for collagen I and III were performed 7 days following each injury. Diabetic wounds displayed decreased collagen at all time points. This was associated with dysregulated collagen production at both the gene and microRNA levels at all time points. Following the final injury, however, diabetic collagen production significantly improved. This appeared to be due to a substantial decrease in both microRNAs as well as an increase in the expression of collagen pathway genes. That dysregulated collagen production progressed throughout the course of wounding suggests that this is one factor contributing to the development of chronic diabetic wounds. Future studies using this model will allow for the determination of other factors that may also contribute to the development and/or persistence of chronic diabetic wounds.

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 cardiac regeneration after fetal myocardial infarction requires cardiac progenitor cell recruitment.

BACKGROUND: In contrast to the adult, fetal sheep consistently regenerate functional myocardium after myocardial infarction. We hypothesize that this regeneration is due to the recruitment of cardiac progenitor cells to the infarct by stromal-derived factor-1α (SDF-1α) and that its competitive inhibition will block the regenerative fetal response. METHODS: A 20% apical infarct was created in adult and fetal sheep by selective permanent coronary artery ligation. Lentiviral overexpression of mutant SDF-1α competitively inhibited SDF-1α in fetal infarcts. Echocardiography was performed to assess left ventricular function and infarct size. Cardiac progenitor cell recruitment and proliferation was assessed in fetal infarcts at 1 month by immunohistochemistry for nkx2.5 and 5-bromo-2-deoxyuridine. RESULTS: Competitive inhibition of SDF-1α converted the regenerative fetal response into a reparative response, similar to the adult. SDF-inhibited fetal infarcts demonstrated significant infarct expansion by echocardiography (p < 0.001) and a significant decrease in the number of nkx2.5+ cells repopulating the infarct (p < 0.001). CONCLUSIONS: The fetal regenerative response to myocardial infarction requires the recruitment of cardiac progenitor cells and is dependent on SDF1α. This novel model of mammalian cardiac regeneration after myocardial infarction provides a powerful tool to better understand cardiac progenitor cell biology and to develop strategies to cardiac regeneration in the adult.

Autophagy is involved in endoplasmic reticulum stress-induced cell death of rat hepatocytes.

BACKGROUND: Both endoplasmic reticulum (ER) stress and autophagy have been shown to display dual roles in cell survival in multiple cell lines. There is a reported but poorly understood link between ER stress, autophagy, and cell death. We hypothesized that autophagy plays a role in ER stress-dependent cell death in rat hepatocytes. MATERIALS AND METHODS: Primary hepatocytes isolated from both lean and obese male Zucker rats were cultured and treated with tunicamycin (TM), tauroursodeoxycholic acid, 3-methyladenine, and wortmannin for 12 h. The ER stress-associated genes glucose-regulated protein 78 and C/EBP homologous protein were examined via quantitative real time polymerase chain reaction. Immunostaining with microtubule-associated protein 1 light chain 3 as well as electron microscopy were used to evaluate autophagy activity. Trypan blue exclusion was used to determine hepatocyte cell viability. RESULTS: In both lean and steatotic hepatocytes, we found that TM induced both C/EBP homologous protein and glucose-regulated protein 78 messenger RNA expression. Cells with increased ER stress were undergoing increased autophagy and had a significant decrease in cell viability. Both tauroursodeoxycholic acid and 3-methyladenine treatments attenuated TM induced ER stress, autophagy, and cell death, whereas wortmannin treatment reduced autophagy and cell death but without changing ER stress. CONCLUSIONS: These data suggest that autophagy is a likely downstream mediator of ER stress-induced cell death in rat hepatocytes. Further exploration of the link between autophagy and ER stress in hepatocyte injury will yield important information that may be leveraged for treatment of liver injuries such as ischemia/reperfusion.

Considering Q fever when working with laboratory sheep.

The sheep (Ovis aries) is somewhat less common than smaller species in laboratory settings, but personnel who work with sheep or in a facility that houses sheep should be aware that certain zoonotic diseases are common in sheep. They should also know how to prevent transmission of zoonotic disease in facilities that house or work with small ruminants. Knowledge of diseases such as query fever (Q fever), which can cause severe human morbidity (and in some cases death), needs to be especially emphasized. In this paper, the author describes potential causes, transmission and manifestations of Q fever in humans and other animals and then discusses strategies for preventing the spread of Q fever.

Sex differences in the pressor response to angiotensin II when the endogenous renin-angiotensin system is blocked.

The present study determined whether there are sex differences in the pressor response to angiotensin II (Ang II) when the endogenous renin-angiotensin system (RAS) is blocked by enalapril (ACEI), and whether this pressor response is changed in the presence of high salt (HS). Telemetry BP was measured in rats treated with ACEI (250 mg/L drinking water) (n=6 to 7/grp), or with ACEI and Ang II (150 ng/kg/min, sc; n=5 to 6/grp), for 3 wk. For the last 2 wk of the study, rats received HS (4% NaCl). MAP was lower in females during baseline (100.8+/-1.1 versus 105.2+/-1.3; P<0.05), and with ACEI the last 3 days on normal salt diet (78.8+/-1.2 versus 88.5+/-0.9; P<0.05), but increased to higher levels than in males on day 6 of Ang II (129.0+/-2.2 versus 117.3+/-2.9; P<0.05). One week of Ang II increased albuminuria in males, but not females, and urinary 8-iso-PGF2alpha (F2-isoP) was not increased in either males or females. MAP was salt-sensitive in both sexes receiving ACEI, but was only salt-sensitive in males with Ang II (129.3+/-3.7 versus 145.1+/-5.7; P<0.05). Albuminuria continued to increase with HS and Ang II in males, but not in females. F2-isoP excretion increased with MAP during the last week of HS and Ang II in males but was independent of MAP in females. With ACEI, MAP in females on normal salt is more responsive to Ang II but is independent of oxidative stress or renal injury. MAP in males is salt-sensitive with Ang II, which may be mediated by oxidative stress and renal injury.

New Topical Agents for Treatment of Partial-thickness Burns in Children: A Review of Published Outcome Studies.

 Evidence-based choices for treating burns in children are not well defined. Skin substitutes and contemporary dressings offer potential advantages over traditional treatment with topical antimicrobial agents in treating partial-thickness burns. Newer treatment modalities may reduce morbidity, financial burdens, and scarring by accelerating healing. Reports of pediatric burn management from 1997 to 2007 were reviewed to compare agent performance with outcome measures such as healing time, pain moderation, cosmetic results, and hospital costs. Transcyte™ (Smith & Nephew, London), Biobrane® (Bertek Pharmaceuticals Inc, Morgantown, WV), beta-glucan collagen, and Mepitel® (Mölnlycke, Göteborg, Sweden) have been reported as superior to silver sulfadiazine (SSD) in achieving faster healing times and decreased pain in pediatric patients. Initial reports describing the outcomes achieved with these new agents indicate that they may offer clinical advantages in the treatment of partial-thickness burns in children. Increased costs of the new products appeared to be offset by decreases in hospital stay, nursing care time and pain medications. The existing literature is not conclusive, and prospective trials with standardized outcome measures are needed to better define the role of these agents. .

Sex hormones and aortic wall remodeling in an arteriovenous fistula.

BACKGROUND: An arteriovenous fistula (AVF) creates high blood flow through the artery and fistula. With this high flow, there is flow-induced remodeling and an increase in diameter, but no intimal hyperplasia. Estrogen has been shown to modify vascular remodeling, decreasing intimal hyperplasia after endothelial injury. OBJECTIVE: These experiments tested the hypothesis that estrogen administration would decrease wall thickness in an AVF model. Because estrogen may decrease wall thickness, we also tested the hypothesis that testosterone would increase wall thickness. METHODS: A fistula was created between the abdominal aorta and the inferior vena cava in Sprague-Dawley rats to generate high blood flow conditions in the aorta. Four groups of female animals were examined: sham, control with AVF ovariectomized (OVX) with AVF and OVX plus testosterone with AVF Four groups of male animals were also examined: sham, control with AVF castrated with AVF and castrated plus estrogen with AVF Five weeks after creation of the AVF, the aortas were collected and fixed; wall thickness was measured both proximal and distal to the AVF. RESULTS: Ovariectomy resulted in a significant decrease in estrogen levels (P < 0.01). Testosterone administration tended to increase testosterone levels in the OVX females, but values did not approach levels observed in the control males. No difference was noted in the proximal wall thickness between the control and the OVX animals. The OVX females receiving testosterone exhibited a significant increase in both proximal and distal wall thickness compared with control females (P < 0.001). In the male animals, there was no significant change in aortic wall thickness in the castrated rats compared with the controls. Estrogen administration in the castrated males resulted in a significant decrease in wall thickness in the proximal and distal aorta (P < 0.05). CONCLUSION: These studies suggest that, in a model of vascular remodeling, estrogen administration decreases wall thickness, and testosterone administration increases wall thickness.

Review of microsurgery and arteriovenous fistulae for hemodialysis.

Patient comorbities, a patient's age and weight, the experience of the surgeon, and state of current vascular access are all factors used in determining the employment of microsurgical techniques to create or salvage an arteriovenous fistula (AVF) for hemodialysis. The aim of this study was to provide an overview of the literature concerning the use of microsurgery for AVFs as the permanent vascular access for both adults and children with end-stage renal disease (ESRD). The patient's overall state of health and long-term survival are always prime considerations in any medical management situation, and any technique that can make these goals more easily attainable by more patients is to be highly valued. Microsurgery for the establishment and repair of AVFs is another useful tool which is available if needed, depending on the unique needs of the patient, to aid the medical community in providing the best possible healthcare.

Evaluation of fructose 1,6 diphosphate for salvage of ischemic gracilis flaps in rats.

Fructose 1, 6 diphosphate (FDP), a metabolic intermediate, provides an alternative mechanism to circumvent the rate-limiting step in the Kreb's cycle. This agent has been observed to prevent the effects of ischemia on heart tissue and kidney function and the effects of endotoxic shock. It has been shown conclusively to minimize the adverse effects of ischemia-reperfusion injury in experimental pedicled skin flaps in animals. The present study was done to evaluate the effect of intra-arterial administration of FDP on salvage of ischemic microvascular transfer of gracilis muscle flaps in rats, with the premise that it might prolong the ischemia time of muscle flaps at room temperature, thus increasing chances of flap survival. Irrigation with FDP did not change the quantitative survival of the flaps, but there was qualitative improvement on histologic evaluation and DNA analysis. Decreased inflammatory damage and DNA fragmentation were seen at the 2.5-hr period. Histologic staining for mitochondrial oxygenation in gracilis muscle also showed increased uptake in the FDP-treated group vs. control at the 2.5-hr ischemia period. Further experiments with different modes of FDP administration should be carried out to identify more effective means of amelioration of flap ischemia.

Rat models of skin wound healing: a review.

Rats have been widely used in the study of skin wound healing and the efficacy of different treatment modalities. This particular animal species is often selected for its availability, low cost, and small size. To define the current use of rat skin wound healing models, this manuscript provides a review of articles published between 2000 and 2003 that chose rats as their research animals. Of the 55 articles reviewed, it was found that 38.2% of the studies used incisional models and 38.2% used excisional models, with some studies using combinations. The majority of the studies (78.2%) used the rat's dorsum as the wound location. Male Sprague Dawley in the 250-300 gram weight range were the most preferred rats. Sodium pentobarbital/pentobarbitone was the most commonly used anesthetic choice. Similarities and differences in the selected experimental conditions are noted and questions are raised with regard to comparability between studies and the ability to transfer the data from the animal model to the human clinical situation. Attempts to compare studies for the advancement of wound healing knowledge are being hampered by the differences found between the studies. Standardization in reporting could facilitate comparisons and may instigate additional research that favors the inevitable comparisons between the studies. Thus, universal reporting requirements need to be developed for animal wound healing studies.

Effect of vascular endothelial growth factor on rat Achilles tendon healing.

This study evaluated the effect of exogenous vascular endothelial growth factor (VEGF) on tendon healing and regulation of other growth factors in a rat Achilles tendon model. Fifty Sprague-Dawley rats were used. In the experimental group, the left Achilles tendon was transected and repaired with the modified Kessler suture technique, and the right Achilles tendon was transected and repaired with resection of plantaris tendon. VEGF, 100 mul (50 mug/ml), was injected into each tendon at the repair site. The same surgical procedures were performed in the control group, with the same volume of saline injected into the repair sites. At intervals of 1, 2, and 4 weeks, the animals were killed and the tendons were harvested and evaluated for tensile strength (1, 2, and 4 weeks) and gene expression (postoperative day 4). At 1 week postoperatively, when plantaris tendon was preserved, the tensile strength of the repaired tendons with VEGF treatment (3.63 +/- 0.62 MPa) was significantly higher than the tensile strength of the repaired tendons with saline treatment (2.20 +/- 0.36 MPa). There was no difference in tensile strength between the two groups without the plantaris tendon support. At 2 weeks postoperatively, the tensile strength was 11.34 +/- 3.89 MPa in the group with VEGF treatment and plantaris tendon preservation, which was significantly higher than the tensile strength in the other groups. There was no significant difference in tensile strength among the groups at 4 weeks postoperatively. The gene expression showed that transforming growth factor-beta in the VEGF-treated tendon was up-regulated in the early stage of tendon healing, whereas expression of platelet-derived growth factor, basic fibroblast growth factor, and insulin-like growth factor-1 was not significantly different among the groups. In conclusion, administration of exogenous VEGF can significantly improve tensile strength early in the course of the rat Achilles tendon healing and was associated with increased expression of transforming growth factor-beta.

Effect of sodium diphenylhydantoin on skin wound healing in rats.

This study evaluated the effect of phenytoin (sodium diphenylhydantoin) on skin wound healing in a rat model. The study was divided into two parts. In part I, 20 mul of phenytoin (10 mg/ml) was subcutaneously injected into the 3-cm dorsal full-thickness incisional wounds of 14 rats on postoperative days 0, 3, and 6. Twelve rats that received saline injections were used as the controls. The skin samples were harvested and tested for tensile strength and histology. An additional 12 rats with the same incisional wounds were tested for chemokine gene expressions. In part II, 20 mul of phenytoin (10 mg/ml) was applied topically once a day on a 4 x 4 cm area of the open dorsal wounds of 10 rats. Saline was applied to the wounds of the 10 control group rats. The wounds were measured weekly. The results showed that the average tensile strength of the phenytoin-treated wound was 0.49 +/- 0.08 MPa compared with the control group at 0.02 +/- 0.01 MPa (p < 0.05). The density ratio of chemokine monocyte chemotactic protein (MCP-1) to beta-actin in the phenytoin-treated group was also significantly higher than in the control group (p < 0.05). Histologic analysis of the phenytoin group showed a large amount of fibroblast proliferation, collagen synthesis, and neovascularization. Phenytoin-treated wounds were also smaller at 1 to 6 weeks postoperatively than the control group wounds. The authors conclude that the administration of phenytoin can promote wound healing and significantly increase MCP-1 expression. Phenytoin-treated wounds showed significant increase in collagen deposition and neovascularization, which resulted in an increased wound tensile strength and accelerated healing of both open and closed wounds.

Small intestinal submucosa in abdominal wall repair after TRAM flap harvesting in a rat model.

The strength of porcine small intestinal submucosa in abdominal wall repair after transverse rectus abdominis myocutaneous flap harvesting was examined in a rat model. Changes in the levels of selected molecular markers of inflammation after small intestinal submucosa implantation were also studied. Eighty-three rats were divided into three groups. In experimental group I, an abdominal wall defect created by removal of the rectus abdominis muscle was repaired with placement of a 1.5 x 5-cm2 patch of small intestinal submucosa. In experimental group II, the muscle defect was repaired with a combination of small intestinal submucosa patch placement and fascial closure. In the control group, the defect was repaired with direct fascial closure. At postoperative times of 3 days, 2 weeks, 1 month, and 2 months, the muscle tissues adjacent to the abdominal wall repair site were subjected to biopsies for assessment of inflammation markers. Full-thickness sections of the abdominal wall from the repair site in each animal were removed for tensile strength testing and histological examinations. The results demonstrated that interleukin-6 and interferon-gamma levels were increased in the two experimental, small intestinal submucosa-treated groups at 3 days and 2 weeks postoperatively. The results of mechanical testing demonstrated that the average tensile strength of the repaired abdominal wall in the repair model with combined small intestinal submucosa placement and fascial repair was significantly greater than the values for repairs with fascial closure or small intestinal submucosa placement alone. The use of small intestinal submucosa placement in combination with fascial repair can significantly improve the strength of the repaired abdominal wall after transverse rectus abdominis myocutaneous flap harvesting.

Role of free radicals in necrosis of skin graft compromised with hematoma.

It has been observed previously that a hematoma affects skin flap survival adversely through free radical action. The current study was undertaken to determine whether similar mechanisms are operative in skin grafts. The experiment was divided into two parts. During part I, 2 x 2 cm2 split-thickness skin grafts (STSGs) were harvested from 18 Fischer rats and were divided randomly into three groups (each consisted of six grafts), and incubated with plasma, blood, and blood plus 70 mg deferoxamine for 48 hours respectively. Tissue samples were assayed for lipoperoxidation (malondialdehyde [MDA]), superoxide dismutase (SOD), and nitric oxide synthase (NOS). During part II, 36 STSGs were harvested and were divided randomly into three groups. The grafts were incubated as in part I for 48 hours. The STSGs were then affixed to the same dimension recipient beds created on the back of 36 inbred rats. Survival was evaluated 7 days postoperatively. The results showed that there was no significant difference in MDA and NOS levels between each incubated graft group in part I. Only the SOD level in both grafts incubated with plasma and blood plus deferoxamine were significantly higher than the grafts over blood alone (p < 0.05). During part II, there was no significant difference of the average STSG survival percentage between the groups incubated with blood and blood plus deferoxamine (35.8 +/- 6.5% and 52.0 +/- 9.5%). The survival percentage of the group incubated with plasma was 81.8 +/- 7.3%, which was significantly higher than the other two groups (p < 0.01). The authors concluded that unlike a distal flap model, the pathological importance of free radicals in survival of the STSG over a hematoma is insignificant. A more likely hypothesis, as suggested by others, is that a hematoma serves as a barrier preventing angiogenesis.