Amyloid beta and impairment in multiple memory systems in older transgenic APP TgCRND8 mice.

The relationship between amyloid beta and cognitive dysfunction in mouse models of Alzheimer's disease has been evaluated predominantly with the spatial reference memory version of the water maze task. However, as Alzheimer's disease encompasses decline in multiple memory systems, it is important to also utilize non-spatial tasks to fully characterize the role of amyloid on behaviour in animal models. We used the TgCRND8 mouse model of Alzheimer's disease to evaluate the effect of amyloid on spatial reference memory, as well as on the non-spatial task of acquisition of conditioned taste aversion, and on the procedural task of swimming to a visible platform. We demonstrate that 8- to 12-month-old TgCRND8 mice are significantly impaired in all three tasks, and that the levels of soluble amyloid beta are significantly correlated with impairment in spatial reference memory, but not with impairment in conditioned taste aversion or swimming to a visible platform. Insoluble fractions of amyloid, which correspond closely to amyloid plaque burden in the brain, are not associated with any behavioural measure. Our study extends the characterization of the model to stages of advanced amyloid pathology and demonstrates that older TgCRND8 mice are impaired in multiple memory systems, including procedural tasks, which are spared at younger ages. The lack of association between amyloid plaques and memory decline supports clinical findings in Alzheimer's patients.

Free-flight encounters between praying mantids (Parasphendale agrionina) and bats (Eptesicus fuscus).

Through staged free-flight encounters between echolocating bats and praying mantids, we examined the effectiveness of two potential predator-evasion behaviors mediated by different sensory modalities: (1) power dive responses triggered by bat echolocation detected by the mantis ultrasound-sensitive auditory system, and (2) ;last-ditch' maneuvers triggered by bat-generated wind detected by the mantis cercal system. Hearing mantids escaped more often than deafened mantids (76% vs 34%, respectively; hearing conveyed 42% advantage). Hearing mantis escape rates decreased when bat attack sequences contained very rapid increases in pulse repetition rates (escape rates <40% for transition slopes >16 p.p.s. 10 ms(-1); escape rates >60% for transition slopes <16 p.p.s. 10 ms(-1)). This suggests that echolocation attack sequences containing very rapid transitions (>16 p.p.s. 10 ms(-1)) could circumvent mantis/insect auditory defenses. However, echolocation attack sequences containing such transitions occurred in only 15% of the trials. Since mantis ultrasound-mediated responses are not 100% effective, cercal-mediated evasive behaviors triggered by bat-generated wind could be beneficial as a backup/secondary system. Although deafened mantids with functioning cerci did not escape more often than deafened mantids with deactivated cerci (35% vs 32%, respectively), bats dropped mantids with functioning cerci twice as frequently as mantids with deactivated cerci. This latter result was not statistically reliable due to small sample sizes, since this study was not designed to fully evaluate this result. It is an interesting observation that warrants further investigation, however, especially since these dropped mantids always survived the encounter.

Plasma amyloid beta protein is elevated in late-onset Alzheimer disease families.

OBJECTIVE: Plasma A beta levels are elevated in early-onset Alzheimer disease (AD) caused by autosomal dominant mutations. Our objective was to determine whether similar genetic elevations exist in late-onset AD (LOAD). METHODS: We measured plasma A beta in first-degree relatives of patients with LOAD in a cross-sectional series and in extended LOAD families. We screened these subjects for pathogenic mutations in early-onset AD genes and determined their ApoE genotypes. RESULTS: Plasma A beta is significantly elevated in the LOAD first-degree relatives in comparison to unrelated controls and married-in spouses. These elevations are not due to ApoE epsilon 4 or pathogenic coding mutations in the known early-onset AD genes. CONCLUSIONS: The findings provide strong evidence for the existence of novel, as yet unknown genetic factors that affect late-onset Alzheimer disease by increasing A beta.

beta-Secretase cleavage of the amyloid precursor protein mediates neuronal apoptosis caused by familial Alzheimer's disease mutations.

The amyloid precursor protein (APP) is cleaved by two enzymes, beta-secretase and gamma-secretase, to generate the pathological amyloid beta (Abeta) peptide. Expression of familial Alzheimer's disease (FAD) mutants of APP in primary neurons causes both intracellular accumulation of the C-terminal beta-secretase cleavage product of APP and increased secretion of Abeta, and eventually results in apoptotic death of the cells. To determine whether either of these two processing products of APP is involved in this apoptotic pathway, we first modeled experimentally the accumulation of the beta-secretase cleavage product in neurons. The C-terminal 100 amino acids (C100) of APP, with and without a signal peptide, was expressed in cells via recombinant herpes simplex virus (HSV) vectors. Both transgene products were targeted to the membrane, and both caused apoptosis in the neurons, implicating the beta-secretase cleavage product of APP in apoptosis caused by FAD APPs. Expression in neurons of a mutant of FAD APP that inhibited beta-secretase cleavage inhibited its ability to cause apoptosis. However, expression in neurons of a mutant of FAD APP that inhibited gamma-secretase cleavage did not inhibit the ability of this mutant to cause apoptosis. These data suggested that the C-terminal beta-secretase cleavage product of APP, but not Abeta, mediates the apoptosis caused by FAD mutants of APP. Consistent with this hypothesis, C31, which is generated from the beta-secretase cleavage product, itself caused neuronal apoptosis. Inhibitors of caspases 3, 6 and 8, but not of caspase 9, inhibited the apoptosis caused by FAD mutants of APP. It may be inferred from these data that beta-secretase cleavage of FAD mutants of APP allows the appropriate caspase access to its site of action to produce C31, which directly causes neuronal apoptosis.

Transcription factor MIZ-1 is regulated via microtubule association.

A synthetic drug, T113242, activates low-density lipoprotein receptor (LDLR) transcription in the presence of sterols. T113242 also covalently binds to beta-tubulin and induces microtubule depolymerization. The myc-interacting zinc finger protein (MIZ-1) associates with microtubules, can bind directly to the LDLR promoter, and can activate LDLR transcription. MIZ-1 also binds to the promoter and activates transcription of other T113242-induced genes such as alpha(2) integrin. Soft X-ray, indirect immunofluorescence, and green fluorescent protein time-lapse microscopy reveal that MIZ-1 is largely cytoplasmic but accumulates in the nuclei of HepG2 cells upon treatment with T113242. Thus, MIZ-1 appears to be regulated by association with microtubules and may activate gene transcription in response to changes in the cytoskeleton.

High throughput screens for the identification of compounds that alter the accumulation of the Alzheimer's amyloid beta peptide (Abeta).

Evidence gathered over the last two decades suggests that beta amyloid (Abeta), the predominant proteinaceous component of senile plaques, plays an early and critical role in the etiology and pathogenesis of Alzheimer's disease (AD). Thus, it is reasonable to hypothesize that compounds capable of reducing the accumulation of Abeta may be of value therapeutically. Additionally, compounds that influence Abeta accumulation may be useful as tools to further dissect the cellular pathways that regulate Abeta production and accumulation. To screen for compounds that affect Abeta levels, we have established high throughput, cell-based assays capable of the sensitive and selective detection of Abeta40 in parallel with the more amyloidogenic form of the peptide, Abeta42. To validate the approach, we examined the effects of several compounds previously identified to influence Abeta accumulation. Analysis of peptide accumulation following treatment with these compounds showed results similar to those previously published. Currently, we are using this assay to screen drugs that have already received FDA approval for the treatment of other diseases and over-the-counter natural product extracts. If compounds such as these can be identified that lower Abeta in the brain, they may represent one of the fastest and most cost effective methods to therapy.

Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP.

JNPL3 transgenic mice expressing a mutant tau protein, which develop neurofibrillary tangles and progressive motor disturbance, were crossed with Tg2576 transgenic mice expressing mutant beta-amyloid precursor protein (APP), thus modulating the APP-Abeta (beta-amyloid peptide) environment. The resulting double mutant (tau/APP) progeny and the Tg2576 parental strain developed Abeta deposits at the same age; however, relative to JNPL3 mice, the double mutants exhibited neurofibrillary tangle pathology that was substantially enhanced in the limbic system and olfactory cortex. These results indicate that either APP or Abeta influences the formation of neurofibrillary tangles. The interaction between Abeta and tau pathologies in these mice supports the hypothesis that a similar interaction occurs in Alzheimer's disease.

Low-vision patients with age-related maculopathy read RSVP faster when word duration varies according to word length.

Normally sighted younger and elder subjects as well as subjects with central visual field loss (CFL) from age-related maculopathy read rapid serial visual presentation (RSVP) text with words presented at a constant rate and at three different rates where word presentation duration varied according to word length. The elder subjects reading sentences foveally read fastest when word duration was constant. The younger group reading random words peripherally read faster at a variable word duration rate. The subjects with CFL read sentences an average of 33% faster when the presentation rate varied with word length. There was a trend for slow readers with CFL to benefit more than fast readers with CFL. We conclude that varying word duration based on word length in rapid serial visual presentation reading would improve reading rates for low-vision patients with CFL.

Modified cotton gauze dressings that selectively absorb neutrophil elastase activity in solution.

Dressings for chronic human wounds have been aimed at protection, removal of exudate, and improved appearance. However since the time of ancient Greece wound care and dressing strategies have primarily relied on empiricism. Recent studies have shown that chronic wounds contain high levels of tissue and cytokine destroying proteases including collagenase and neutrophil elastase. Therefore we sought to develop an effective wound dressing that could absorb elastase through affinity sequestration. Cotton gauze was modified by oxidation, phosphorylation, and sulfonation to enhance elastase affinity by ionic or active site uptake. Type VII absorbent cotton gauze was oxidized to dialdehyde cotton which was subsequently converted in part to the bisulfite addition product. Gauze preparations were also phosphorylated and carboxymethylated. Modified cotton gauzes were compared with untreated gauze for reduction of elastase activity in buffered saline. Solutions of elastase that were soaked in oxidized, sulfonated, and phosphorylated cotton gauze showed reduced elastase activity. The initial velocities (v(o)) and turnover rates of elastase showed significant decreases compared with solutions taken from untreated gauze. The reduction in enzyme activity with dialdehyde cotton gauze was confirmed in solution by determining elastase inhibition with dialdehyde starch. The dialdehyde cotton gauze also decreased elastase activity in human wound fluid in a dose response relation based on weight of gauze per volume of wound fluid. Absorbency, pH, air permeability and strength properties of the modified gauze were also compared with untreated cotton gauze. This report shows the effect of reducing elastase activity in solution with cotton containing aldehydic or negatively charged cellulose fibers that may be applicable to treatment modalities in chronic wounds.

High resolution protein localization using soft X-ray microscopy.

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 microm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive "water window", where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (lambda = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.

Characterization of recombinant, soluble beta-secretase from an insect cell expression system.

The beta-site amyloid precursor protein-cleaving enzyme (BACE) cleaves the amyloid precursor protein to produce the N terminus of the amyloid beta peptide, a major component of the plaques found in the brains of Alzheimer's disease patients. Sequence analysis of BACE indicates that the protein contains the consensus sequences found in most known aspartyl proteases, but otherwise has only modest homology with aspartyl proteases of known three-dimensional structure (i.e., pepsin, renin, or cathepsin D). Because BACE has been shown to be one of the two proteolytic activities responsible for the production of the Abeta peptide, this enzyme is a prime target for the design of therapeutic agents aimed at reducing Abeta for the treatment of Alzheimer's disease. Toward this ultimate goal, we have expressed a recombinant, truncated human BACE in a Drosophila melanogaster S2 cell expression system to generate high levels of secreted BACE protein. The protein was convenient to purify and was enzymatically active and specific for cleaving the beta-secretase site of human APP, as demonstrated with soluble APP as the substrate in novel sandwich enzyme-linked immunosorbent assay and Western blot assays. Further kinetic analysis revealed no catalytic differences between this recombinant, secreted BACE, and brain BACE. Both showed a strong preference for substrates that contained the Swedish mutation, where NL is substituted for KM immediately upstream of the cleavage site, relative to the wild-type sequence, and both showed the same extent of inhibition by a peptide-based inhibitor. The capability to produce large quantities of BACE enzyme will facilitate protein structure determination and inhibitor development efforts that may lead to the evolution of useful Alzheimer's disease treatments.

Reduction of Abeta accumulation in the Tg2576 animal model of Alzheimer's disease after oral administration of the phosphatidyl-inositol kinase inhibitor wortmannin.

The abnormal accumulation of the amyloid beta protein (Abeta) has been implicated as an early and critical event in the etiology and pathogenesis of Alzheimer's disease (AD). Compounds that reduce Abeta accumulation may therefore be useful therapeutically. In cell-based screens we detected a significant reduction in Abeta concentration after treatment with the phosphatidylinositol kinase inhibitors wortmannin and LY294002. To determine the effect of this class of compounds on in vivo Abeta accumulation, we administered wortmannin to the Tg2576 mouse model of AD. Oral administration of wortmannin over four months resulted in a significant, non-overlapping 40%-50% reduction in the number of senile plaques, one of the pathological hallmarks of AD. Sandwich ELISA analysis of formic acid extractable Abeta in the brain of treated animals indicates that both Abeta40 and the longer, more amyloidogenic form of the peptide, Abeta42, were significantly reduced. These data provide the first direct evidence that compounds identified by their ability to reduce Abeta concentration in vitro can reduce Abeta accumulation and deposition in the brain, thus establishing a basic paradigm for the identification and evaluation of additional compounds that lower Abeta accumulation.

Proinflammatory cytokines differentially regulate hyaluronan synthase isoforms in fetal and adult fibroblasts.

BACKGROUND/PURPOSE: Fetal wound healing is a relatively scarless process that occurs in an hyaluronan-rich environment. Understanding the regulation of hyaluronan expression may provide insight into the process of fetal repair. Therefore, the purpose of this study was to compare the regulation of hyaluronan and hyaluronan synthase transcripts by the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in human adult and fetal fibroblasts. METHODS: Hyaluronan deposited in the medium of untreated fibroblasts or fibroblasts treated with either IL-1beta or TNF-alpha was determined by an assay utilizing iodine I 125-hyaluronan binding protein. HAS transcript levels were compared in using a ribonuclease protection assay. RESULTS: IL-1beta induced an increase in hyaluronan accumulation by both fetal and adult fibroblasts. In contrast, TNF-alpha induced higher levels of hyaluronan only in fetal fibroblasts. HAS-2 and HAS-3 transcript levels were constitutively expressed by both fetal and adult fibroblasts. Proinflammatory cytokines induced a differential increase in HAS-1 and HAS-3 transcript levels. CONCLUSIONS: Differential regulation was observed in hyaluronan accumulation and for HAS transcript levels in fetal and adult dermal fibroblasts. The muted response of fetal fibroblasts to cytokines may be relevant to the minimal inflammation associated with fetal repair.

Myofibroblast induction with transforming growth factor-beta1 and -beta3 in cutaneous fetal excisional wounds.

BACKGROUND/PURPOSE: In a noncontractile fetal rabbit model, the authors recently have shown the induction of excisional wound contraction with sustained-release cellulose implants formulated with transforming growth factor (TGF)-beta. The purpose of this study was to test the hypothesis that the excisional wound contraction in this model is associated with the induction of myofibroblasts in the surrounding dermis, demonstrated by the presence of alpha-smooth muscle actin. METHODS: Cellulose discs were formulated with either 1.0 microg of TGF-beta1 (n = 6); 1.0 microg of TGF-beta3 (n = 9); 10 microg of TGF-beta3 (n = 6); or their carrier protein, bovine serum albumin (BSA; n = 9), for sustained-release over 5 days. Each disc was implanted into a subcutaneous pocket on the back of a fetal New Zealand White rabbit in utero on day 24 of gestation (term, 31 days). A full-thickness, 3-mm excisional wound (7.4 mm2) was then made next to the implanted cellulose disc. All fetuses were harvested at 3 days. The amount of alpha-smooth muscle (SM) actin in the dermis around the implants and wounds was determined using immunohistochemical techniques. RESULTS: Excisional wounds exposed to 1.0 microg of TGF-beta1 (5.6+/-2.0 mm2), 1.0 microg of TGF-beta3 (6.9+/-1.0 mm2), and 10 microg of TGF-beta3 (2.7+/-1.0 mm2) were significantly smaller when compared with the BSA control group (12.8+/-1.1 mm2; P<.05). Furthermore, there was a significant increase in staining for alpha-SM actin in the TGF-beta1 (1.8+/-0.5) and 10 microg TGF-beta3 (2.8+/-0.2) groups in comparison with the scant staining in the BSA control group (0.5+/-0.2; P<.05). CONCLUSIONS: TGF-beta1 and -beta3 induce alpha-SM actin and contraction of cutaneous excisional wounds in a fetal noncontractile model. This model of inducible cutaneous excisional wound contraction may be useful in further determining the role of the myofibroblast in wound contraction and the physiology underlying this poorly understood aspect of wound healing.

Antisense-induced reduction of presenilin 1 expression selectively increases the production of amyloid beta42 in transfected cells.

Autosomal dominant mutations in the presenilin 1 (PS1) gene are associated with familial, early-onset Alzheimer's disease. Although the pathogenic mechanism of these mutations is unclear, their common feature is that they lead to an increased concentration of amyloid beta-peptide (Abeta) 42 in the plasma of early-onset patients, in the conditioned media of transfected cells, and in the brains of transgenic mice that overexpress mutant PS1. To address the mechanism(s) by which the pathogenic PS1 mutations increase Abeta42, we constructed human cell lines expressing a doxycyclin (dox)-inducible antisense PS1 RNA and measured its effects on the levels of PS1, amyloid precursor protein (APP), and Abeta. In time course experiments, we observed a statistically significant (p = 0.0038) more than twofold elevation in secreted Abeta42 as early as 12 days after addition of dox. This correlated with an 80% decrease in the 46-kDa PS1 holoprotein and a 30% decrease in the 26-kDa N-terminal fragment (NTF). Furthermore, there was a significant fivefold (p = 0.002) increase in Abeta42 after 14-day dox treatment; this correlated with a >90% decrease in PS1 holoprotein and 60% decrease in NTF. At no time point did we observe significant changes in Abeta40, APP holoprotein, presenilin 2, or tubulin. Ten days after the removal of dox, we observed a return to constitutive levels for Abeta42, PS1 holoprotein, and NTF. These results suggest that in human cell lines, the reduction of normal PS1 activity results in the increased production of Abeta42. Furthermore, our results are consistent with a loss of function or dominant negative mechanism for the pathogenic PS1 mutations.

Structure, development, and evolution of insect auditory systems.

This paper provides an overview of insect peripheral auditory systems focusing on tympanate ears (pressure detectors) and emphasizing research during the last 15 years. The theme throughout is the evolution of hearing in insects. Ears have appeared independently no fewer than 19 times in the class Insecta and are located on various thoracic and abdominal body segments, on legs, on wings, and on mouth parts. All have fundamentally similar structures-a tympanum backed by a tracheal sac and a tympanal chordotonal organ-though they vary widely in size, ancillary structures, and number of chordotonal sensilla. Novel ears have recently been discovered in praying mantids, two families of beetles, and two families of flies. The tachinid flies are especially notable because they use a previously unknown mechanism for sound localization. Developmental and comparative studies have identified the evolutionary precursors of the tympanal chordotonal organs in several insects; they are uniformly chordotonal proprioceptors. Tympanate species fall into clusters determined by which of the embryologically defined chordotonal organ groups in each body segment served as precursor for the tympanal organ. This suggests that the many appearances of hearing could arise from changes in a small number of developmental modules. The nature of those developmental changes that lead to a functional insect ear is not yet known.

Method to analyze collagenase and gelatinase activity by fibroblasts in culture.

The net amount of collagen produced and deposited by fibroblasts in cell culture is determined by the rate of collagen synthesis as well as the rate of collagen degradation. Although collagen synthesis can be analyzed by several techniques, it is more difficult to measure collagen degradation. Breakdown of collagen depends upon the activity of a family of structurally and catalytically related mammalian enzymes termed matrix metalloproteinases (MMPs). Interstitial collagenase (MMP1) initiates the cleavage of fibrillar collagen, whereas gelatinases (MMP2 and MMP9) digest the denatured collagen fragments. A method has been developed to quantitate the activity of collagenase (MMP1) and gelatinase (MMP9) in conditioned medium from fibroblast cell cultures. The assay, which uses the fluorogenic substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-AlaLys(Nma)NH2, is technically simple and amenable to high throughput analysis. Addition of specific inhibitors of the metalloproteinases allows for simultaneous measurement of both collagenase and gelatinase activity.

Glycosylphosphatidylinositol-anchored proteins play an important role in the biogenesis of the Alzheimer's amyloid beta-protein.

The Alzheimer's amyloid protein (Abeta) is released from the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase. beta-Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta) and an Abeta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release Abeta. Alternative cleavage of the APP by alpha-secretase at Abeta16/17 releases the secreted derivative sAPPalpha. In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Abeta40 and Abeta42 in the medium but had no effect on sAPPalpha. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta40, Abeta42, and sAPPbeta than its parental line (GPI+). When this parental line was treated with PI-PLC, Abeta40, Abeta42, and sAPPbeta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta-secretase activity and Abeta secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.

TGF-beta1 alters the healing of cutaneous fetal excisional wounds.

BACKGROUND/PURPOSE: In a number of species, fetal wound healing differs from the adult in the absence of inflammation, fibrosis, scar formation, and excisional wound contraction. The lack of inflammation also may explain the relative absence of any cytokine levels at the wound site, such as transforming growth factor (TGF)-beta, and therefore the unique characteristics of fetal wound healing. The authors hypothesized that exogenous TGF-beta1 would induce contraction, inflammation, fibrosis, and scar formation in cutaneous excisional wounds in the fetal rabbit. METHODS: Cellulose discs (3 mm in diameter) were formulated with either 1.0 microg TGF-beta1 (n = 6) or bovine serum albumin (BSA; n = 7), as a control, for sustained-release over 3 days. Each disc was implanted into the subcutaneous tissue on the backs of fetal New Zealand White Rabbits in utero on day 24 of gestation (term, 31 days). A full-thickness, 3-mm excisional wound (7.4 mm2) was then made next to the implanted cellulose disc. All wounds were harvested 3 days later. RESULTS: At harvest, the excisional wounds in the TGF-beta1 group had contracted (5.6 +/- 2.0 mm2), whereas those in the control group had expanded (13.5 +/- 1.2 mm2, P< .01). The surrounding dermis in the TGF-beta1 group had 16.3 inflammatory cells per grid block compared with 12.4 cells in the control group (not significant). In addition, a greater amount of fibrosis was induced by the TGF-beta1 implant (1.7 +/- 0.3) than the control implant (0.4 +/- 0.2) on a scale of 0 to 3, P < .01. In situ hybridization analysis showed an increase in procollagen type 1alpha1 gene expression in the surrounding dermis of the TGF-beta1 group (36.7 +/- 3.6 grains per grid block) compared with the control group (7.1 +/- 0.9 grains per grid block, P < .001). CONCLUSIONS: These results demonstrate that the cytokine TGF-beta1 can induce fetal excisional wounds to contract, stimulate fibrosis, and increase procollagen type 1alpha1 gene expression. These findings further suggest that the absence of TGF-beta1 atthe wound site may be responsible in part for the lack of a postnatal healing response.

MMP-8 is the predominant collagenase in healing wounds and nonhealing ulcers.

BACKGROUND: The initial cleavage of collagen by collagenase represents the rate-limiting step in the degradation of this central extracellular matrix protein. Chronic nonhealing ulcers, especially pressure ulcers, typically contain elevated levels of collagenolytic activity. However, there have been no detailed attempts to identify the source of these collagenases and their activity either in normal healing wounds or in chronic nonhealing ulcers. MATERIALS AND METHODS: Levels of the matrix metalloproteinases, MMP-1 and MMP-8, and the tissue inhibitor of matrix metalloproteinases, TIMP-1, were measured in fluids and tissues of healing human wounds and nonhealing ulcers by ELISA. Relative MMP-1 and MMP-8 levels were also analyzed by substrate preference in a functional assay. RESULTS: The patterns of the collagenases MMP-1 and MMP-8 in healing wounds were distinct, with MMP-8 appearing in significantly greater amounts than MMP-1. Chronic nonhealing ulcers were characterized by significantly higher levels of MMP-1 and MMP-8, and lower levels of TIMP-1, than in healing wounds. Levels of both MMP-1 and MMP-8 varied greatly in chronic ulcers, although MMP-8 was always the predominant collagenase present in these wounds. Interestingly, these collagenases were present almost exclusively in their inactive forms in healing wounds, whereas nonhealing ulcers possessed significant levels of the active forms of these enzymes. CONCLUSIONS: These results clearly demonstrate that the neutrophil-derived MMP-8 is the predominant collagenase present in normal healing wounds and suggest that overexpression and activation of this collagenase may be involved in the pathogenesis of nonhealing chronic ulcers. In addition, excessive collagenolytic activity in chronic ulcers is made possible, partly because of the reduced levels of the inhibitor, TIMP-1.