Hepatic Wound Research Articles

Studies on mRNA expression of basic fibroblast growth factor in wound healing for wound age determination.

We investigated the mRNA expression of basic fibroblast growth factor (bFGF) for wound age determination during dermal, cerebral, hepatic and renal wound healing in mice. The bFGF mRNA expression in the injured skin peaked at 1 h and was detected in epidermal cells, fibroblasts, endothelial cells and neutrophils. In the injured cerebrum the expression increased from 1 h and peaked at 48 h. In the intact cerebrum, bFGF was detected exclusively in the endothelial cells, whereas it was also detected in astrocytes during wound healing. Time-dependent expression of bFGF mRNA in skin and cerebrum was considered to be useful for wound age determination. On the other hand it was suggested that bFGF mRNA in astrocytes could be a vital sign of the acute phase. In hepatic and renal injuries, however, bFGF mRNA expression increased slightly in endothelial cells at 24 h, in neutrophils of the liver and in the glomeruli of the kidneys.

Expression of transmembrane 4 superfamily (TM4SF) proteins and their role in hepatic stellate cell motility and wound healing migration.

Migration of activated hepatic stellate cells (HSC) is a key event in the progression of liver fibrosis. Little is known about transmembrane proteins involved in HSC motility. Tetraspanins (TM4SF) have been implicated in cell development, differentiation, motility and tumor cell invasion. We evaluated the expression and function of four TM4SF, namely CD9, CD81, CD63 and CD151, and their involvement in HSC migration, adhesion, and proliferation. All TM4SF investigated were highly expressed at the human HSC surface with different patterns of intracellular distribution. Monoclonal antibodies directed against the four TM4SF inhibited HSC migration induced by extracellular matrix proteins in both wound healing and haptotaxis assays. This inhibition was independent of the ECM substrates employed (collagen type I or IV, laminin), and was comparable to that obtained by incubating the cells with an anti-beta1 blocking mAb. Importantly, cell adhesion was unaffected by the incubation with the same antibodies. Co-immunoprecipitation studies revealed different patterns of association between the four TM4SF studied and beta1 integrin. Finally, anti-TM4SF antibodies did not affect HSC growth. These findings provide the first characterization of tetraspanins expression and of their role in HSC migration, a key event in liver tissue wound healing and fibrogenesis.

Effects of endothelins on hepatic stellate cell synthesis of endothelin-1 during hepatic wound healing.

Endothelin-1 production is increased after liver injury and the subsequent wounding response. Further, endothelin-1 has prominent effects on hepatic stellate cells (key effectors of the hepatic wounding response), including on collagen synthesis, proliferation, and expression of smooth muscle proteins. We tested the hypothesis that endothelins (ETs) may regulate endothelin-1 production during hepatic wounding, and have investigated potential mechanisms underlying this process. Studies were performed on isolated stellate cells from normal and injured livers; in addition, potential autocrine effects of ET were assessed in vivo using an ET receptor antagonist in a model of liver injury. In stellate cells isolated from either normal or injured rat livers, ET receptor stimulation with endothelin-3 or sarafotoxin S6C (preferential ET(B) agonists) caused a dose-dependent increase in endothelin-1 production. Additionally, administration of a mixed ET antagonist in vivo during injury led to reduced stellate cell production of endothelin-1. The mechanism by which ETs stimulated endothelin-1 in this system appeared to be through upregulation of ET converting enzyme-1 (which converts precursor ET to mature peptide), rather than by modulation of precursor endothelin-1. We conclude that during liver injury and wound healing, stellate cell endothelin-1 production is, at least partially, stimulated by ETs via autocrine mechanisms that occur at the level of ET converting enzyme-1.

JunD Regulates Transcription of the Tissue Inhibitor of Metalloproteinases-1 and Interleukin-6 Genes in Activated Hepatic Stellate Cells

Activation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype is the pivotal event in hepatic wound healing and fibrosis. Rat HSCs activated in vitro express JunD, Fra2, and FosB as the predominant AP-1 DNA-binding proteins, and all three associate with an AP-1 sequence that is essential for activity of the tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter. In this study, we used expression vectors for wild-type, dominant-negative, and forced homodimeric (Jun/eb1 chimeric factors) forms of JunD and other Fos and Jun proteins to determine the requirement for JunD in the transcriptional regulation of the TIMP-1 and interleukin-6 (IL-6) genes. JunD activity was required for TIMP-1 gene promoter activity, whereas overexpression of Fra2 or FosB caused a repression of promoter activity. The ability of homodimeric JunD/eb1 to elevate TIMP-1 promoter activity supports a role for JunD homodimers as the major AP-1-dependent transactivators of the TIMP-1 gene. IL-6 promoter activity was induced upon activation of HSCs and also required JunD activity; however, expression of JunD/eb1 homodimers resulted in transcriptional repression. Mutagenesis of the IL-6 promoter showed that an AP-1 DNA-binding site previously reported to be an activator of transcription in fibroblasts functions as a suppressor of promoter activity in HSCs. We conclude that JunD activates IL-6 gene transcription as a heterodimer and operates at an alternative DNA-binding site in the promoter. The relevance of these findings to events occurring in the injured liver was addressed by showing that AP-1 DNA-binding complexes are induced during HSC activation and contain JunD as the predominant Jun family protein. JunD is therefore an important transcriptional regulator of genes responsive to Jun homo- and heterodimers in activated HSCs.

Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells.

The tissue repair response to hypoxic stimuli during wound healing includes enhanced production of angiogenic factors, such as vascular endothelial growth factor (VEGF). Hepatic stellate cells are oxygen-sensing cells, capable of producing VEGF. We hypothesized that hypoxia-stimulated signaling in activated stellate cells mediate VEGF secretion during liver injury. The specific aim was to evaluate the effect of hypoxia on the gene expression of VEGF in HSC-T6 cells, an immortalized rat hepatic stellate cell line, and in rat primary cultures of stellate cells. Hypoxic induction of VEGF mRNA was dose- and time-dependent. The hypoxic stimulation of VEGF messenger RNA (mRNA) correlated with the secretion of VEGF protein in conditioned media by hypoxic T6 cells. S-Nitroso-N-acetyl-D, L-penicillamine (SNAP), a nitric oxide (NO) donor, and desferrioxamine (DFx) and cobalt chloride, mimics of cellular hypoxia, similarly stimulated VEGF mRNA expression and secretion. Four previously described splice variants of the VEGF mRNA (VEGF-120, 144, 164, 188) were detected in both normoxic- or hypoxic-activated stellate cells. There was differential expression of the VEGF receptors, Flt-1 and Flk-1, in hypoxic T6 cells. Hypoxic conditions selectively stimulated Flt-1 mRNA expression, whereas Flk-1 mRNA remained unchanged. Hypoxic induction of VEGF was also demonstrated in primary stellate cell cultures and after in vivo injury. Hypoxia stimulates cell signaling in stellate cells, culminating in the rapid induction of VEGF and Flt-1 mRNA expression and VEGF secretion. The hypoxic induction of VEGF is mimicked by NO and may be of mechanistic importance in the pathogenesis of hepatic wound healing and hepatocarcinogenesis.

Regulation of Endothelin-1 Synthesis by Endothelin-converting Enzyme-1 during Wound Healing

Endothelin-1 (ET-1) is involved in the pathogenesis of a number of diseases, including wound healing. In cirrhosis, the wounding response of the liver, circulating ET-1 levels are elevated; moreover, ET-1 has potent effects on hepatic stellate cells, the key effectors of cirrhosis. In this study, we have examined the regulatory role of ECE-1, a critical enzyme involved in ET-1 synthesis, in the two major cellular sources of hepatic ET-1. ET-1 release from normal hepatic endothelial cells was 25-fold higher than that from normal stellate cells. However, after liver injury, ET-1 release was increased in stellate cells but markedly decreased in endothelial cells. The two major isoforms of ECE-1, ECE-1alpha/1beta, made up 80% and 20%, respectively, of total ECE-1 in both stellate and endothelial cells. Following liver injury, ECE-1alpha mRNA was decreased by 44.2% in stellate cells, and by 16.1% in endothelial cells. ECE-1beta mRNA expression remained unchanged after injury. In contrast to ECE-1 mRNA, ECE-1 protein expression was increased by 43.9% in stellate cells but decreased in endothelial cells, while relative ECE-1 enzymatic activity was unchanged. In mRNA stability experiments, the half-life of ECE-1alpha mRNA in normal stellate cells was 13 h compared with 38 h in cells from injured livers. Thus, during hepatic wound healing, differential regulation of ECE-1 mRNA and protein appears to be critical in controlling ET-1 production.

Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing.

Homology PCR has been used to identify receptor tyrosine kinases (RTKs) expressed during activation of rat hepatic stellate cells, the key fibrogenic mesenchymal element in the liver. Partial cDNAs encoding several RTKs were cloned from stellate cells activated in vivo, including those of Flt-1, Flk-1, c-met, PDGFR, and Tyro10/DDR2. RNAse protection from cells activated in vivo demonstrated biphasic induction of flt-1 and flk-1 mRNAs, receptors for vascular endothelial growth factor (VEGF). Culture-activation of stellate cells was associated with increased [125I]VEGF binding and Flt-1 and Flk-1 receptor protein. Induction of VEGF binding sites correlated with an 2.5-fold increase in DNA synthesis in response to VEGF, but only if cells were activated by growth on collagen 1, whereas cells maintained in a quiescent state on a basement membrane-like substratum (EHS matrix) were nonproliferative. In both stellate and endothelial cells VEGF-induced mitogenesis was augmented by co-incubation with basic fibroblast growth factor (bFGF), a cytokine with known synergy with VEGF. These findings suggest that the cellular targets of VEGF in liver may not be confined to sinusoidal endothelial cells, and that VEGF responses reflect combined effects on both hepatic stellate cells and sinusoidal endothelium.

Tamponamento das lesões hepáticas transfixantes com colágeno tipo I

Nos últimos seis anos temos utilizado o balão de SANGESTAKEN-BLACKMORE para o tamponamento de lesões transfixantes hepáticas. Para substituir este corpo estranho, consideramos o uso de colágeno tipo I produzido na UNICAMP, o qual pode ser injetado e moldado ocupando toda a área lesada e o trajeto anfractuoso ou irregular. Desta forma, pode-se conseguir um tamponamento completo, coibindo a hemorragia e sustando a perda de bile. O colágeno será posteriormente reabsorvido não funcionando como corpo estranho. Vinte cães foram submetidos a lesão transfixante na região central do fígado, procurando simular uma lesão produzida por um projétil de arma de fogo. Quatro destes animais, não receberam tratamento e foram a óbito por choque hemorrágico. Em todos os outros 16 animais, aplicou-se o colágeno tipo I em toda a extensão do túnel formado pela lesão, através de injeção do material. Foram realizadas avaliações clínicas no pós-operatório, sendo os cães sacrificados, em grupo de quatro, aos quatro, sete, 15 e 30 dias de pós-operatório. O estudo macroscópico e o microscópico, tanto à microscopia óptica quanto à de polarização, revelou uma formação cicatricial homogênea, com regeneração hepática intensa, precoce e organizada, a partir do 7º dia de pós-operatório. Conclui-se que a aplicação do colágeno tipo I, nestes tipos de ferimentos, são de aplicação simples e segura.

Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses.

Hepatic fibrosis represents the generalized response of the liver to injury and is characterized by excessive deposition of extracellular matrix. The cellular basis of this process is complex and involves interplay of many factors, of which cytokines are prominent. We have identified divergent fibrosing responses to injury among mouse strains and taken advantage of these differences to examine and contrast T helper (Th)-derived cytokines during fibrogenesis. Liver injury was induced with carbon tetrachloride, fibrosis was quantitated, and Th1/Th2 cytokine mRNAs measured. Liver injury in BALB/c mice resulted in severe fibrosis, whereas C57BL/6 mice developed comparatively minimal fibrosis. Fibrogenesis was significantly modified in T and B cell-deficient BALB/c and C57BL/6 severe combined immunodeficient (SCID) mice compared with wild-type counterparts, suggesting a role of Th subsets. Fibrogenic BALB/c mice exhibited a Th2 response during the wounding response, whereas C57BL/6 mice displayed a Th1 response, suggesting that hepatic fibrosis is influenced by different T helper subsets. Moreover, mice lacking interferon gamma, which default to the Th2 cytokine pathway, exhibited more pronounced fibrotic lesions than did wild-type animals. Finally, shifting of the Th2 response toward a Th1 response by treatment with neutralizing anti-interleukin 4 or with interferon gamma itself ameliorated fibrosis in BALB/c mice. These data support a role for immune modulation of hepatic fibrosis and suggest that Th cytokine subsets can modulate the fibrotic response to injury.

C-17 TREATMENT OF TRANSFIXING HEPATIC WOUNDS WITH A INTRA-HEPATIC BALLOON

Mantovani, M.; Muraro, CAS; F, A. Concon°; Silveira, HJV; Fraqa, G P Author Information

Nutritional Needs of the Acutely III with Acute Wounds

The purpose of nutritional support in the critically ill is to blunt the effects of the stress response, to preserve musculature and organ function, and to promote hepatic protein synthesis, tissue regeneration, and wound healing. Adequate nutritional support is vital to the patient with acute wounds. This article addresses the body's response to physiologic stress and reviews means of supporting nutritional status and the substrates used to maximize nutritional therapy. The critical care nurse will have a better appreciation for how and when to implement nutritional therapy, appropriate nutrients, and methods for monitoring nutritional status.

Clostridial septic shock following an isolated, hepatic gunshot wound

Clostridial septic shock following an isolated, hepatic gunshot wound

Diagnostic evaluation and surgical management of hydatid disease of the liver.

Human echinococcosis is still endemic in some areas of the world, including Mediterranean countries. Because there is no effective medical therapy, surgery remains the principal mode of treatment. A consecutive of 132 patients operated on for liver hydatid disease between January 1977 and February 1993 were analyzed. There were 60 men (45.4%) and 72 women (54.6%) aged 31 to 88 years (mean 56 years). The right lobe of the liver was affected in 68 cases (51.5%), the left lobe in 31 cases (23.5%), and both lobes in 14 cases (10.6%); there were multiple liver cysts in 7 cases (5.3%), concomitant cysts in other parenchymal organs in 4 cases (3.0%), and disseminated intraabdominal hydatid disease in 8 cases (6.1%). Clinical symptomatology consisted of abdominal pain, fever, jaundice, urticaria, and an abdominal mass. Preoperative diagnosis was established using imaging studies: plain abdominal films, ultrasonography, computed tomography, and serologic tests. Three patients (2.3%) underwent simple closure without drainage, 7 patients (5.3%) cyst excision, 4 patients (3.0%) marsupialization, 1 patient (0.8%) left lateral segmentectomy, 15 patients (11.3%) external drainage, 69 patients (52.3%) omentoplasty, and 33 patients (25.0%) combinations of procedures. Postoperative morbidity was low and consisted of hepatic abscess development, wound infection, bowel obstruction, and biliary leaks. Six patients (4.5%) had recurrent disease. One patient died during the postoperative period because of septic complications. Among the surgical techniques we used, excision of the cyst (when feasible) and omentoplasty produced the lowest complication rates and the best clinical results.

GUNSHOT WOUNDS TO THE RIGHT THORACOABDOMEN

Dissatisified with our unnecessary laparotomy rate in patients with gunshot wounds (GSWs) to the right thoracoabdomen (RTA), a prospective study was designed to test the hypothesis that hemodynamically stable patients without peritonitis could be managed without a surgical procedure. From 1990 through 1993, 13 consecutive patients with a GSW between the right nipple, costal margin, right posterior axillary line, and anterior midline were studied. No patient had or developed more than local wound tenderness. All patients had a right hemothorax treated with a chest tube. Computed tomographic (CT) scanning of the RTA was performed within 8 hours of admission in 12 of the 13 patients, and the following injuries were noted: pulmonary contusion (12), hepatic laceration (seven), spinal cord transection (two), and a renal laceration (one). Follow-up CT scans, 3 to 14 days after injury, in six of the seven patients with hepatic wounds showed partial or complete resolution of the injury in all. In one patient, an associated renal injury was unchanged on the follow-up CT scan. Mean length of hospitalization for the 11 patients who did not have an injury to the spinal cord was 5.1 days (3-8 days). Complications included atelectasis (four), a small persistent pneumothorax (two), and pneumonia (one).(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of transforming growth factor beta receptors of rat lipocytes during the hepatic wound healing response. Enhanced binding and reduced gene expression accompany cellular activation in culture and in vivo.

Activation of lipocytes, characterized by increased proliferation and fibrogenesis, is a central feature of the hepatic wound healing response. We have examined whether modulation of receptors for transforming growth factor beta (TGF-beta) contributes to the fibrogenic behavior of activated lipocytes. Isolated lipocytes were maintained in a quiescent state by culturing the cells in suspension, where they displayed minimal specific binding for TGF-beta 1 and only a small amount of type III (betaglycan) receptor by affinity labeling. In contrast, lipocytes activated by growth on uncoated plastic displayed saturable binding of TGF-beta 1 (Kd = 28 pM, 7,730 receptors/cell), and receptors types I, II, and III. Binding activity in quiescent and activated cells correlated with responsiveness to TGF-beta 1; TGF-beta 1 induced cellular fibronectin mRNA expression only in activated and not quiescent cells. Despite the absence of binding in quiescent cells, type II receptor was detectable by immunoblot. By RNase protection assay, mRNAs for receptor types II and III were greater in quiescent than activated cells. In freshly isolated lipocytes from animals with liver injury caused by the administration of carbon tetrachloride, a rapid but transient increase in mRNA for receptor types I (approximately 3.2-fold), II (approximately 1.5-fold), and III (approximately 3-fold) was observed with peaks at 12 h for type I receptor, 1 h for type II receptor, and 6 h for type III receptor; mRNA induction was followed by down-regulation for all receptors. The modest changes in mRNAs compared with marked alterations in binding activity during mesenchymal cell activation suggest that TGF-beta receptors may be regulated in vivo in part by a post-translational mechanism.

The role of fibronectin in tumor implantation at surgical sites.

Fibronectins are a family of glycoproteins with modular functional domains. They mediate cell-cell and cell-matrix interactions which are important in embryogenesis, wound healing, metastasis and other processes. We present data on the influence of fibronectin on wound implantation of a murine mammary carcinoma line, TA3Ha. Fibronectin used in these studies was derived from bovine plasma, human serum, human foreskin fibroblasts, and mouse embryo cultures. TA3Ha cells rarely form tumors in the liver of syngeneic mice when injected intravenously but after hepatic wedge resection, 45% (107/240) of the mice develop tumors in the hepatic wound. Wound implantation is markedly reduced when the cells are pre-exposed to 200 micrograms/ml bovine plasma fibronectin (13%, P = 0.007), human serum fibronectin (0%, P = 0.02), human cellular fibronectin (0%, P = 0.02), or mouse cellular fibronectin (0%, P = 0.04). Lung colonization is also reduced by these fibronectins. These effects are not due to a cytotoxic action of fibronectin, since intraperitoneally injected fibronectin-treated cells form ascites tumor as effectively as do control untreated cells. Local application of a solution containing 0.25 mg/ml mouse cellular fibronectin to the hepatic wound reduces the frequency of tumor implantation from 45% to 5% (1/21, P = 0.001). No tumor implantation inhibition is seen when only suspending medium or albumin in suspending medium is used. The mechanism by which topical application of fibronectin reduces hepatic wound implantation of tumor cells is unclear, but this finding raises an exciting possibility of preventing local recurrence of cancer.

Inhibition of tumor implantation at sites of trauma by Arg-Gly-Asp containing proteins and peptides

We report on the inhibition of wound implantation by TA3Ha mammary carcinoma cells by Arg-Gly-Asp containing proteins and peptides using a hepatic wedge resection model. Intravenously injected TA3Ha cells rarely form tumor in the liver of syngeneic mice, but after hepatic wedge resection, 45% (107/240) of the mice develop tumors in the hepatic wound. Hepatic wound implantation is significantly (P = 0.01) inhibited by pretreating the cells with whole mouse plasma, but not with fibrinogen-depleted plasma or serum. Tumor inhibition is also achieved by pretreatment of cells with fibrinogen (P = 0.05-0.0004), fibronectin (P = 0.007) and laminin, but not by albumin. The active domain appears to be the RGDS sequence since the deca- and tetrapeptides containing RGDS inhibit wound implantation (P less than 0.05). However, the tetrapeptide Arg-Gly-Glu-Ser has no such activity. None of these agents affects ascites tumor formation by the intraperitoneally injected cells, suggesting that anchorage independent growth of cells is not affected. We propose that proteins and peptides containing RGD occupy the binding sites and prevent the cells from interacting with cell adhesion proteins in healing wounds. Proteins and/or peptides containing RGD may be useful for preventing local recurrence in postsurgical cancer patients.

Hepatic trauma: experience with 135 consecutive liver injuries (1982?1989) and arguments for conservative surgery

Most hepatic traumas are easily cured, but the series of 135 consecutive hepatic wounds reported in the present paper is unusual in that the patients were specifically recruited from among patients subjected to neurosurgical or spinal operations, 25% of whom sustained severe hepatic injuries (classes IV and V) as a result of the surgery. The postoperative mortality was analyzed according to such potentially predictive factors as severity of the hepatic wound, the concomitant extraabdominal lesions, the initial shock, and the kind of surgical treatment. The statistical comparison of the factors affecting the results was analyzed by the Chi-square test. The postoperative mortality rate was 24.4% (33 deaths). This mortality rate is evidently related to the severity of the hepatic lesions and to the frequent associated lesions. The 14 deaths from benign and moderate hepatic injuries were due to concomitant lesions. Among the 19 deaths from severe lesions, 12 were directly related to the severity of the hepatic injury and 7 to associated wounds. Complications directly related to the hepatic trauma occurred in 39 cases with 16 deaths. In general, conservative surgical treatment can be performed with quite low mortality. Among the patients who require hepatic resection one of two dies of hemorrhage or coagulopathy. Among conservative procedures, perihepatic packing has proved to be efficient and safe. If perioperative cholangiography has excluded any leak from a major bilde duct, septic complications are rare. Therefore, the surgical treatment of hepatic trauma should be as conservative as possible, because this can stop hemorrhage and decrease the risk of coagulopathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of tumor implantation at sites of trauma by plasminogen activators

The authors report on the influence of plasminogen activators (PA) on implantation of TA3Ha mammary tumor cells in the healing hepatic wounds of syngeneic strain A mice. Intravenously injected TA3Ha cells, although they rarely metastasize to the liver, formed tumors in the hepatic wounds of a significant percent (42%, P less than 0.0001) of mice. The frequency of tumor formation declined as the interval between surgery and tumor cell inoculation was increased. Furthermore, preexposure of cells to fibrinogen, fibronectin, laminin, or peptides containing the arginine-glycine-aspartic acid-serine residues dramatically reduced the frequency of tumor formation in the hepatic wounds. These results indicate that TA3Ha cells interact with fibrinogen-related proteins in the wound to aid their attachment and growth. Because these proteins are susceptible to digestion by plasmin, PA were used in this study to examine whether administration of these drugs to the mice would modulate tumor formation in the liver wounds. Among the PA tested, human plasmin B-chain-streptokinase complex (B-SK) and recombinant tissue plasminogen activator (t-PA) inhibited tumor implantation in a dose-related manner. Administration of 900 units (U) of B-SK or 3300 U of t-PA per mouse reduced the frequency of tumor formation from 42% to 0% (P = 0.02) and 11% (P = 0.02), respectively. The B-SK was complexed with p-nitrophenyl-p-guanidinobenzoate; it did not activate the plasminogen or inhibit tumor formation in the hepatic wounds. Although urokinase activated the plasminogen, it did not inhibit tumor implantation in the hepatic wound. Heparin, an anticoagulant that prevents conversion of fibrinogen to fibrin without being fibrinolytic, had no influence on tumor formation in the hepatic wounds. The PA can generate plasmin that digests the cell attachment proteins in wounds and consequently inhibits tumor cell attachment.

Warning

Two cases of severe hypotension following the use of fibrin glue for hemostasis in hepatic injuries are reported. A systemic reaction to bovine thrombin via large venous lacerations is suspected. A preliminary animal study supports this hypothesis. Caution is advised in the use of fibrin glue for hemostasis in deep hepatic wounds.