Roy W. Tarnuzzer

Biochemical analysis of acute and chronic wound environments.

The process of wound healing involves a complex interaction between numerous cell types, extracellular matrix molecules, and soluble mediators including growth factors and cytokines. This complex milieu is under active investigation for the purposes of beginning to understand how this environment regulates tissue repair. Quantitation of growth factors, cytokines, and matrix metalloproteinases within surgical wound fluids may help to elucidate this regulatory network, not only in noncomplicated wound healing but also in pathologic lesions such as chronic ulcers.

Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans

Abstract.Despite the association of increasing age with chronic wound-healing disorders and an impaired rate of healing of acute cutaneous wounds, the role of matrix metalloproteinases (MMPs) is unknown. To determine the spatial and temporal patterns and activities of MMP-1, -2, -3 and -9, 132 healthy humans aged between 19 and 96 years underwent 4-mm punch biopsies followed by wound excision between day 1 and day 180 post-wounding. Wounds showed an age-related increase in MMP-2 and MMP-9 immunostaining from day 3; this was associated with degradation of gelatin as shown by zymograms and with increased proteinase activity as shown by azocoll assays. Distinct spatial localisations for each MMP were observed: MMP-2 was found in epidermal structures; MMP-9 was observed in inflammatory cells up to day 21; MMP-1 was localised to keratinocytes at the wound margin. Normal old skin showed pro-MMP-2 bands on zymography and increased MMP-2 immunostaining. These results indicate that: (1) intrinsic ageing is associated with the up-regulation of MMPs previously associated with chronic wound healing; (2) wound-tissue proteinases are essentially active up to day 21 postwounding; and (3) intrinsic ageing may predispose to tissue breakdown disorders because of MMP-2 up-regulation in normal skin.

Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 proteins and mRNA

Proteolysis is an essential component of wound healing but, if uncontrolled, it may lead to degradation of the neo‐matrix and a delay in wound repair. Despite numerous reports of impaired wound healing associated with increasing age, the control of proteolysis is completely unknown. Tissue inhibitor of matrix metalloproteinases (TIMP)‐1 and ‐2 inhibit the activity of matrix metalloproteinases and the pattern of regulation of these molecules determines in part the spatial and temporal regulation of proteolytic activity. This study reports on TIMP‐1 and ‐2 protein localization using immunocytochemistry in healing wounds of healthy subjects of different ages from day 1 to 6 months post‐wounding, and has quantified the mRNA levels for both inhibitors using reverse transcriptase‐polymerase chain reaction (RT‐PCR). TIMP‐1 and TIMP‐2 proteins are up‐regulated from 24 h post‐wounding, with a decrease in staining intensity by day 7 for TIMP‐2 and by day 14 for TIMP‐1. Steady‐state mRNA levels for both TIMPs were significantly greater in normal young skin than in aged skin. In the young, there was a significant increase in mRNA expression for TIMP‐1 and ‐2 by day 3 post‐wounding, which decreased by day 14 and had returned to basal levels at day 21. In the wounds of the aged subjects, basal levels were observed for TIMP‐1 and ‐2 at all time‐points. These results suggest that intrinsic cutaneous ageing is associated with reduced levels of TIMP mRNA both in normal skin and during acute wound repair. These levels may be instrumental in dermal tissue breakdown in normal skin, retarded wound healing, and the predisposition of the elderly to chronic wound healing states.

Expression of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases in mesothelial cells and their regulation by transforming growth factor-beta1.

Tissue injury and pelvic inflammation often results in peritoneal scar tissue formation. The objective of this study was to determine whether mesothelial cells which line the peritoneal cavity express matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), and if their expression is regulated by transforming growth factor‐β1, a key regulator of tissue fibrosis. For this purpose we used Met‐5A cells, a cell line derived from human normal mesothelial cells, and for comparative analysis we used U‐937 cells, a human monocytic/macrophage cell line. The cells were treated with transforming growth factor‐β1 (1 ng/ml) for various time periods and the levels of MMP and TIMP mRNA and protein expression were determined using quantitative reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. The results indicate that the mesothelial cells and macrophages express MMP‐1 (collagenase‐1), MMP‐3 (stromelysin‐1), TIMP‐1 and TIMP‐2 mRNA and protein at various levels, with significantly higher TIMPs than MMPs, and higher MMP‐1 than MMP‐3 (p < 0.001). The mesothelial cells express significantly less MMP‐1, higher MMP‐ 3 and similar levels of TIMP mRNA compared to macrophages. In a time‐dependent manner, treatment of the mesothelial cells with transforming growth factor‐β1 resulted in a significant decrease in the expression of MMP‐1, while increasing the expression of TIMP‐1 mRNA (p = 0.05). In contrast, MMP‐3 and TIMP‐2 expression was unaffected in mesothelial cells and in macrophages, compared to untreated controls. There was a significant increase in secreted MMP‐1 and TIMP‐2 by mesothelial cells following transforming growth factor‐β1 treatments in a time‐dependent manner (p = 0.05 and p = 0.01), without affecting the secretion of these proteins by macrophages. A major portion of MMP‐1 in the culture conditioned media of both cell types was found in complex with TIMP‐1. The ratios of MMP‐1/TIMPs production were significantly higher than MMP‐3/TIMPs in mesothelial cells and macrophages, and progressively decreased following transforming growth factor‐β1 treatments (p < 0.05). In conclusion, these results indicate that mesothelial cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by transforming growth factor‐β1, a mechanism that in part may influence the outcome of peritoneal tissue repair and adhesion formation.

Expression of IGF-I, IGF-I receptor and IGF binding proteins - 1, -2, -3, -4 and -5 in human atherectomy specimens

The molecular and cellular processes that induce rapid atherosclerotic plaque progression in patients with unstable angina and initiate restenosis following coronary interventional procedures are uncertain. We examined primary (de novo) and restenotic lesions retrieved at the time of directional coronary atherectomy for expression of insulin-like-growth factor-I (IGF-I). IGF-I receptor, and five IGF binding proteins (IGFBPs), IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 in smooth muscle cells (SMCs) using colloidal gold immunocytochemistry. IGF-1, its receptor and binding proteins were not detected in SMCs of normal coronary arteries. IGF-I localized primarily in synthetic smooth muscle cells (sSMCs) in both de novo and restenotic plaques. IGF-I receptor localized on sSMCs and their processes and colocalized with IGF-I. Although morphometric analysis of IGF-I and IGF-I receptor immunoreactivity in sSMCs of de novo and restenotic lesions showed comparable levels of IGF-I (3.2 +/- 1.0 and 2.9 +/- 0.9, respectively). IGF-I receptor was significantly higher in de novo lesions as compared to restenotic lesions (10.7 +/- 2.5 and 4.2 +/- 1.3, P < 0.05, respectively). IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-5 localized in the cytoplasm of sSMCs and in the extracellular matrix. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) performed on de novo atherectomy specimens identified mRNA for IGF-I, IGF-I receptor, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5 levels and detected mRNA for IGFBP-3. The expression of IGF-I, IGF-I receptor, and IGFBPs in atherectomy plaques suggests that the development of coronary obstructive lesions may be a result of changes in the IGF system.

Matrix metalloproteinase expression in human retinal microvascular cells.

The degree of hyperglycemia correlates with the development of diabetic retinopathy. We investigated the effect of glucose on the expression of matrix metalloproteinase (MMP)-2 and MMP-9 (72-kDa and 92-kDa type IV collagenases, respectively) by human retinal microvascular endothelial cells (HRECs). Cultured HRECs from nondiabetic and diabetic donors were exposed to 5 or 30 mmol/l glucose. Using gelatin zymography, conditioned medium (CM) from all cultures revealed a gelatinolytic band migrating at 65 kDa (representing the proform of MMP-2 that runs at 72 kDa under reducing conditions). This band was unchanged by glucose exposure or the disease state of the donors. CM from nondiabetic HREC cultures demonstrated an additional proteolytic activity migrating at 90 kDa when cells were exposed to 30 mmol/l glucose, but not when they were exposed to 5 mmol/l glucose. This same activity was seen in CM from HREC cultures of diabetic origin in the presence of both 5 and 30 mmol/l glucose. Western analysis confirmed the identity of the 65-kDa band as MMP-2. The anomalous activity at 90 kDa was identified as MMP-2 associated and co-migrating with a fibronectin fragment. Competition-based reverse transcription-polymerase chain reaction revealed that nondiabetic and diabetic HRECs expressed constitutively mRNA for MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and fibronectin. After exposure to 5 or 30 mmol/l glucose, no changes were detected in mRNA levels in MMP-2 or MMP-9, their inhibitors TIMP-1 and TIMP-2, or fibronectin in either nondiabetic or diabetic HREC cultures. These results support the notion that modulation of MMP function by extracellular matrix components occurs in response to glucose and may be relevant to the development of diabetic retinopathy.

Insulin-like growth factor: Receptor and binding proteins in human retinal endothelial cell cultures of diabetic and non-diabetic origin

Human retinal endothelial cell (HREC) cultures of diabetic and non-diabetic origin were examined for the production of insulin-like growth factor I (IGF-I), IGF-I receptor and IGF-binding proteins (IGFBPs) using colloidal gold quantitative immunocytochemistry and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR). The levels of immunoreactivity for IGF-I receptor and for four IGFBPs (IGFBP-1, -2, -3 and -5) were significantly increased in diabetic HREC cultures. Moreover, diabetic HREC cultures showed significantly less immunoreactivity for IGF-I and for IGFBP-4 as compared to non-diabetic HREC cultures. Message levels for IGF-I decreased two-fold in diabetic HREC and correlated with protein levels. Message levels for IGFBP-1, -2 and -5 increased 1.5-, 1.7- and 1.6-fold, respectively, in diabetic HREC and correlated with protein levels. However, the protein levels for IGF-I receptor and IGFBP-3 and -4 did not correlate with mRNA levels. There were no differences in mRNA levels for IGF-I receptor and IGFBP-3 and -4 between diabetic and non-diabetic HREC cultures, suggesting a post-transcriptional regulation of IGF-I receptor and the two IGFBPs. The net effect, however, supports enhanced IGF-I action in HREC cultures of diabetic origin which is an important cellular event in diabetic retinopathy.

EXTRACELLULAR-MATRIX GENE EXPRESSION DURING MOUSE SUBMANDIBULAR GLAND DEVELOPMENT

Early morphogenesis of mouse submandibular glands begins on late day 11 of fetal development when the epithelium begins to bud from the surrounding mandibular mesenchyme. Using total RNA collected from fetal BALB/c submandibular glands, steady-state levels of mRNA expression for extracellular matrix molecules were measured using quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR). By comparing the PCR amplification products of both the cellular mRNA and a synthetic template, pMATRIX, it was possible to measure the direct expression of collagens alpha2(I), alpha1(III), alpha1(IV), fibronectin, laminin B2, elastin and lysyl oxidase genes. There was an observed trend for an increasing concentration of collagen alpha2(I), collagen alpha1(III) and lysyl oxidase mRNA molecules per cell on day 16 of development. The relative abundance of elastin mRNA was detectable only on day 16. Fibronectin and laminin B2 were more constitutively present but had their highest copy number per cell on day 16. The presence of extracellular-matrix protein was confirmed by immunohistochemistry using day-16 fetal glands and adult glands. With the construction of the pMATRIX supertemplate and the advent of quantitative, competitive RT-PCR technology, it has been possible to measure small changes in the steady-state concentrations for extracellular-matrix mRNA during salivary gland development.

Heterochromia after pediatric cataract surgery

PURPOSE Changes in iris color have been noted anecdotally after cataract surgery in infants, but they have not been studied systematically. The mechanism for these iris color changes has not previously been reported in the biomedical literature. METHODS Photographs were taken of both eyes of 15 children and 11 rhesus monkeys who had undergone unilateral cataract surgery. Masked examiners reviewed the photographs and compared the iris color of the eyes that were operated on with the eyes that were not operated on. Between 4 and 6 weeks postoperatively, the level of prostaglandin F(2alpha) in the aqueous humor (n = 4) and vitreous humor (n = 2) was measured in both the operated and nonoperated eyes of 4 monkeys that had undergone a neonatal lensectomy during the first 5 days of life. RESULTS Thirteen of 15 children had a darker iris color in the operated eye in relation to the nonoperated (control) eye. Four of 11 monkeys had a uniformly darker iris in the operated eye; the other 7 monkeys had regional darkening or patches of darker iris in the eye that was operated on. The prostaglandin F(2alpha) levels in neonatal monkeys were higher in the aqueous humor and in the vitreous humor of the operated eye in relation to the nonoperated eye. CONCLUSION In some children, cataract surgery is associated with a darkening of the iris color in the operated eye. We speculate that this darkening results from an exuberant prostaglandin release stimulated by the cataract surgery and may occur through the same or a similar mechanism by which latanoprost causes the darkening of iris color.

Expression of alpha-smooth muscle actin, TGF-β1 and TGF-β type II receptor during connective tissue contraction

SummaryClosure of rat mesenteric perforation is considered to occur by connective tissue contraction, a process that has been shown to be stimulated by transforming growth factor-β1. In the present study, we assessed the expression of alpha-smooth muscle actin during closure by quantitative-reverse transcription-polymerase chain reaction and in situ hybridization. The expression of transforming growth factor-β1 and transforming growth factor-β type II receptor was also estimated in mesenteric membranes and free peritoneal cells after wounding.A larger expression of alpha-smooth muscle actin was seen around the wound edges compared to unwounded tissue. Both alpha-smooth muscle actin and transforming growth factor-β type II receptor were expressed during Days 0, 3, 5, 7, and 10. The expression of alpha-smooth muscle actin on Day 5 was >100 times higher than on Day 0. Transforming growth factor-β1 was expressed in both membranes and free peritoneal cells of unoperated control animals but down-regulated after wounding, a finding that has not been reported previously. It reappeared on Days 7 and 10 in free peritoneal cells but not in perforated membranes.The enhanced expression of alpha-smooth muscle actin and down-regulation of transforming growth factor-β1 expression after wounding appears to be important phenomena in tissue contraction and repair.