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Notes: Chronic abdominal sepsis is associated with impaired tissue repair. Treatment of burn patients with growth hormone results in improved healing of skin graft donor sites. The goal of this study was to determine whether administration of growth hormone could attenuate the inhibitory effects of sepsis on cutaneous wound healing. Four groups of male Sprague Dawley rats were studied: control, control + growth hormone, sepsis, and sepsis + growth hormone. Sepsis was caused by implantation of a bacterial focus in the peritoneal cavity. Control animals underwent sham laparotomy, and polyvinyl alcohol sponge implants were placed in subdermal pockets in all animals. Saline or growth hormone (400 μg) was injected subcutaneously every 12 hours. On day 5, the incisional wounds and polyvinyl alcohol sponge implants were harvested. The breaking strength of abdominal incisions was measured. Granulation tissue penetration and quality were determined by scoring polyvinyl alcohol sponge implant histology from 1 to 4 in a blinded fashion. Collagen deposition in polyvinyl alcohol sponge implants was quantitated by hydroxyproline assay. Septic mortality was not altered by growth hormone administration. Septic animals showed a reduction in food consumption for 2 days after surgery (p 〈 0.05 vs. controls), which was not affected by growth hormone administration. The breaking strength of incisional wounds and hydroxyproline content of polyvinyl alcohol sponge implants was reduced in septic rats (p 〈 0.001 vs. controls) but administration of growth hormone for 5 days did not improve breaking strength or collagen deposition in either group. We conclude that the administration of growth hormone for 5 days did not improve collagen deposition or breaking strength in cutaneous wounds from control or septic animals. The results suggest that growth hormone treatment is unlikely to improve tissue repair in sepsis-induced catabolic illness.

Notes: The release of mast cell granules is commonly associated with inflammation and fibrosis. However, does direct communication between mast cells and fibroblasts through gap junction intercellular communication (GJIC) occur? Fibroblast populated collagen lattice (FPCL) cast with mast cells show enhanced lattice contraction. Do released granules or GJIC between mast cells and fibroblasts promote enhanced lattice contraction? Mast cells preloaded with a fluorescent dye that readily passes through gap junctions were cast in FPCL. Dye passed from mast cells into fibroblasts within these cocultured mast cell-FPCLs. Fatty acid amide hydrolase inhibitor blocks the breakdown of oleamide, which is a potent endogenous inhibitor of GJIC. GJIC was blocked for 3 days when mast cells were pulsed for 3 hours with fatty acid amide hydrolase inhibitor. Mast cells pretreated with fatty acid amide hydrolase inhibitor cast in cocultured mast cell-FPCLs failed to enhance cocultured lattice contraction. Mast cell-FPCLs made with mouse fibroblasts unable to generate GJIC failed to show enhanced lattice contraction. Degranulated mast cells were equal to intact mast cells at enhancing cocultured mast cell-FPCL contraction. The supernatant from degranulated mast cells had no effect upon FPCL contraction. Therefore, enhanced mast cell-FPCL contraction appears to be independent of mast cell granules, but dependent upon GJIC between fibroblasts and mast cells. We speculate that mast cell–fibroblast GJIC may play a role in fibrosis.

Notes: The participation of fibroblasts in wound repair is a coordinated effort requiring sequential cellular modulations to behavior including migration (entering), proliferation (increasing cell numbers), synthesis (depositing a collagen matrix), remodeling (organizing collagen), transformation into myofibroblasts, apoptosis, and elimination. Disruptions in that orderly sequence of behaviors will alter repair. Insights into controlling wound repair have focused on soluble factors such as cytokines and growth factors. Here we examine the direct communications between coupled cells through gap junctional intercellular communications. Molecules of less than 1000 MW pass directly between cells through gated gap junction channels. Sugars, amino acids, and oxygen, as well as second messengers such as cAMP, inositol phosphates, and calcium can pass directly between coupled cells. Does gap junctional intercellular communication affect fibroblast phenotype progression in granulation tissue maturation? In rats gap junctional intercellular communication uncouplers heptanol and endosulfan were injected daily into polyvinyl alcohol sponge implants. At 7 days, uncoupler-treated implants had capsules with increased fibroblast density, reduced cell penetration into the sponge, and diminished numbers of myofibroblasts. By polarized light, the uncouplers reduced the deposition and organization of collagen and thereby disrupted the coordinated phenotypic changes seen in fibroblasts during the repair process. It is proposed that gap junctional intercellular communication is critical for fibroblast progression from migratory cell to apoptosis as granulation tissue matures into scar. (WOUND REP REG 2003;11:481–489)

Notes: Wound healing requires fibroblast migration, synthesis of new extracellular matrix, and organization of that matrix, all of which depend upon myosin ATPase activation and subsequent cytoplasmic actin-myosin contraction. Myosin ATPase activity is optimized by phosphorylation of myosin light chain at serine 19. Several different signaling pathways can perform that phosphorylation, the focus here is calcium saturated calmodulin dependent -myosin light chain kinase (CaM-MLCK). It is proposed that CaM-MLCK phosphorylation of myosin light chain and subsequent myosin ATPase activation affects granulation tissue fibroblast behavior and contributes to wound contraction. Myosin ATPase activity generates actin-myosin contraction within fibroblasts. Myosin ATPase activity is involved in ATP-induced cell contraction, the generation of focal adhesions, fibroblast migration, fibroblast populated collagen lattice (FPCL) contraction, and wound contraction. The MLCK inhibitors ML-9 and ML-7 inhibited ATP-induced cell contraction, fibroblast migration, FA formation, and FPCL contraction. The calmodulin inhibitors W7 and fluphenazine blocked rat open wound contraction. In addition, fluphenazine delayed re-epithelialization. These findings support the idea that fibroblast CaM-MLCK activity is essential for tissue repair. We speculate that inhibition of CaM-MLCK may reduce or prevent detrimental fibrotic contracture.

Notes: Systemic ingestion of vanadate, a nonspecific inhibitor of tyrosine phosphatases, doubles wound breaking strength, enhances the packing of collagen fibers, and prevents the appearance of myofibroblasts in granulation tissue. Will the local application of vanadate mimic the systemic effects? Pairs of polyvinyl alcohol sponges, each with a central reservoir and attached injection port, were subcutaneously implanted in rats. Daily, one implant received 0.2 ml of saline and the other received 0.2 ml of 0.03 mM vanadate in saline. On day 7, harvested sponges had equivalent wet weights. The vanadate-treated sponges had fibroblasts separated by connective tissue, with a more intense birefringence of the collagen fibers. Transmission electron microscopy showed collagen more uniformly packed in the vanadate treated sponges where collagen fibers were equally spaced and had equal diameters. By immunohistology, myofibroblasts, defined by the expression of α-smooth muscle actin within stress fibers, were absent in vanadate-treated granulation tissue. The expression of α-smooth muscle actin was restricted to smooth muscle cells of blood vessels. Controls had densely packed α-smooth muscle actin staining myofibroblasts, weak birefringence, and randomly spaced collagen fibers with irregular diameters. We conclude that the local application of vanadate prevents the appearance of myofibroblasts and optimizes the organization of collagen fibers in developing granulation tissue. (WOUND REP REG 2003;11:204–212)

Notes: There are numerous causes for slow or delayed wound healing. Because slowly healing wounds are often inflamed, we quantitated the inflammatory chemokine, interleukin-8, produced by slowly healing human burn wounds and compared this to interleukin-8 from healed wounds and normal intact skin. Interleukin-8 levels were increased significantly in unhealed wounds (19.7 ng/ml) compared to healed wounds (7.7 ng/ml) or normal skin (5.7 ng/ml). Interleukin-8 in these ranges was added to adult human keratinocytes and fibroblasts. Interleukin-8 significantly decreased keratinocyte replication but had no effect on fibroblast replication. The rate or final degree of fibroblast populated collagen lattice contraction was inhibited at interleukin-8 concentrations between 10 and 30 ng/ml, but not altered at concentrations below 10 ng/ml and above 100 ng/ml. The concurrent application of indomethacin at 10 μg/ml reversed this interleukin-8 induced inhibition. Interleukin-8 inhibited myosin ATPase activity, apparently by reducing the phosphorylation of nonmuscle myosin light chain. We conclude that elevated levels of interleukin-8 may be found during delayed healing, and these elevated interleukin-8 levels may directly contribute to retarded wound closure.

Notes: Introduction: Immediate reinitiation of atrial tachyarrhythmia (IRAT) is an important cause of failure to maintain sinus rhythm. IRAT prevention by overdrive pacing has not been evaluated in a prospective randomized trial. Methods and Results: Patients with a DDDRP pacemaker offering temporary atrial overdrive pacing after AT termination (Post Mode Switching Overdrive Pacing [PMOP]) were enrolled into the prospective PIRAT (Prevention of IRAT) trial if paroxysmal AT episodes occurred after implantation. PMOP was randomly activated (120 beats/min for 2 min) or inactive. After 3 months, device memory was interrogated, symptoms and quality of life assessed, and patients crossed over to the alternative treatment arm for another 3 months. Primary study endpoint was the number of AT episodes; secondary endpoints were the cumulative time in AT (AT burden), percentage of AT episodes with IRAT, symptoms, and quality of life with PMOP active versus inactive. In 37 patients (21 men; 69 ± 9 years), there was no difference in the median number of AT episodes (0.37 vs 0.34 per day), AT burden (both 1%), percentage of episodes with IRAT (30%vs 28%), symptoms, and quality of life during PMOP off versus on. With PMOP active, 29% of 439 ATs restarted during and 18% before PMOP intervention. The PMOP-induced rate increase appeared to be associated with IRAT in 9% of AT episodes. Conclusion: Automatic overdrive pacing after AT termination did not prevent IRAT, mainly due to insufficient overdrive suppression even at 120 beats/min and the delay between AT termination and PMOP intervention. (J Cardiovasc Electrophysiol, Vol. 14, pp. 954-959, September 2003)

Notes: AF in Congestive Heart Failure. Atrial fibrillation (AF) and congestive heart failure (CHF) are two increasingly common cardiac disorders with a growing prevalence in the overall population. Improved treatment of acute medical conditions has increased the incidence of these cardiac disorders. AF and CHF have similar epidemiologic characteristics and adversely affect quality of life and life expectancy of affected patients. CHF predisposes to AF, and AF may worsen the prognosis of CHF. The relevant literature was intensively reviewed with emphasis on aspects at the intersection of both disease sets. Recent advances in basic research have provided a more in-depth view of changes promoting the occurrence of AF in CHF. Data from clinical trials have provided means to improve medical treatment of AF. Precautions must be taken for specific CHF-related side effects, such as torsades de pointes tachycardia, when treating AF. The specific electrophysiologic basis of AF associated with CHF may provide targets for improved treatment modalities. New treatment approaches, both pharmacologic and nonpharmacologic, as well as the results of ongoing controlled clinical studies are likely to greatly alter AF therapy over the next 5 to 10 years in patients with CHF.

Notes: Studies of metabolism of the Alzheimer amyloid precursor protein (APP) have focused much recent attention on the biology of juxta- and intra-membranous proteases. Release or ‘shedding’ of the large APP ectodomain can occur via one of two competing pathways, the α- and β-secretase pathways, that are distinguished both by subcellular site of proteolysis and by site of cleavage within APP. The α-secretase pathway cleaves within the amyloidogenic Aβ domain of APP, precluding the formation of toxic amyloid aggregates. The relative utilization of the α- and β-secretase pathways is controlled by the activation of certain protein phosphorylation signal transduction pathways including protein kinase C (PKC) and extracellular signal regulated protein kinase [ERK/mitogen-activated protein kinase (MAP kinase)], although the relevant substrates for phosphorylation remain obscure. Because of their apparent ability to decrease the risk for Alzheimer disease, the effects of statins (HMG CoA reductase inhibitors) on APP metabolism were studied. Statin treatment induced an APP processing phenocopy of PKC or ERK activation, raising the possibility that statin effects on APP processing might involve protein phosphorylation. In cultured neuroblastoma cells transfected with human Swedish mutant APP, atorvastatin stimulated the release of α-secretase-released, soluble APP (sAPPα). However, statin-induced stimulation of sAPPα release was not antagonized by inhibitors of either PKC or ERK, or by the co-expression of a dominant negative isoform of ERK (dnERK), indicating that PKC and ERK do not play key roles in mediating the effect of atorvastatin on sAPPα secretion. These results suggest that statins may regulate α-secretase activity either by altering the biophysical properties of plasma membranes or by modulating the function of as-yet unidentified protein kinases that respond to either cholesterol or to some intermediate in the cholesterol metabolic pathway. A ‘phospho-proteomic’ analysis of N2a cells with and without statin treatment was performed, revealing changes in the phosphorylation state of several protein kinases plausibly related to APP processing. A systematic evaluation of the possible role of these protein kinases in statin-regulated APP ectodomain shedding is underway.