May J. Reed

Differential expression of SPARC and thrombospondin 1 in wound repair: immunolocalization and in situ hybridization.

SPARC and thrombospondin 1 (TSP-1) are secreted glycoproteins expressed by similar types of cells in culture and in tissues. To compare these two proteins in vivo, we analyzed the differential expression of SPARC and TSP-1 during wound repair. Full-thickness incision wounds were made in rats and biopsied at 12 hr-14 days. Antibodies against SPARC revealed an increased proportion of immunoreactive fibroblastic cells at the wound edge at 3 days with maximal numbers at 7 days. In situ hybridization for SPARC produced results consistent with those of immunohistochemistry. With combined immunohistochemistry and in situ hybridization, some of the macrophages at the wound edge expressed SPARC mRNA. In contrast, immunoreactivity for TSP-1 was extracellular; expression at the wound edge was noted at 12 hr and was maximal at 1-2 days. TSP-1 mRNA was found in the thrombus, but not at the wound edge. In conclusion, SPARC and TSP-1 have contrasting roles during wound healing. SPARC expression from the middle through late stages of repair was consistent with its previously proposed functions in remodeling; in contrast, the transient expression of TSP-1 early in repair might facilitate the action of other proteins in recruitment and/or proliferation of cells in the healing wound.

SPARC-null mice exhibit accelerated cutaneous wound closure.

Expression of SPARC (secreted protein acidic and rich in cysteine; osteonectin, BM-40), an extracellular matrix (ECM) associated protein, is coincident with matrix remodeling. To further identify the functions of SPARC in vivo, we have made excisional wounds on the dorsa of SPARC-null and wild-type mice and monitored closure over time. A significant decrease in the size of the SPARC-null wounds, in comparison to that of wild-type, was observed at Day 4 and was maximal at Day 7. Although substantial differences in the percentage of proliferating cells were not apparent in SPARC-null relative to wild-type wounds, primary cultures of SPARC-null dermal fibroblasts displayed accelerated migration, relative to wild-type fibroblasts, in wound assays in vitro. Although the expression of collagen I mRNA in wounds, as measured by in situ hybridization (ISH), was not significantly different in SPARC-null vs wild-type mice, the collagen content of unwounded skin appeared to be substantially lower in the SPARC-null animals. By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of wild-type skin. Moreover, we found an inverse correlation between the efficiency of collagen gel contraction by dermal fibroblasts and the concentration of collagen within the gel itself. We propose that the accelerated wound closure seen in SPARC-null dermis results from its decreased collagen content, a condition contributing to enhanced contractibility.

Cleavage of the matricellular protein SPARC by matrix metalloproteinase 3 produces polypeptides that influence angiogenesis.

SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu2+-binding sequence KHGK) exhibited a biphasic effect on [3H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca2+-binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu2+-binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.

Impaired Angiogenesis in Aging Is Associated with Alterations in Vessel Density, Matrix Composition, Inflammatory Response, and Growth Factor Expression

It is generally accepted that angiogenesis is delayed in aging. To define the effects of age on the neovascular response, polyvinyl alcohol sponges were implanted SC in young (6–8 months old, n = 11) and aged (23-25 months old, n = 13) mice and sampled at 14 and 19 days. Angiogenic invasion was significantly delayed in aged mice at 14d relative to young at 14d (% area of invasion 9.0 ± 3.7 vs 19.0 ± 5.6; p = 0.02). Although microvessel morphology and basement membrane composition were similar between the age groups, a significant decrease in capillary density was noted in aged tissues at 14d (7.5 ± 4.1) and 19d (12.1 ± 2.8) relative to young at 14d (18.7 ± 2.3) (p<0.01 A14d vs Y14d). In comparison to young at 14d, the inflammatory response was decreased by 43 ± 2.9% and 36 ± 7.8% in aged mice at 14d and 19d, respectively. Tissues of aged mice showed less newly deposited collagen. There was a lack of expression of transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) in aged mice at 14d (0.63 ± 0.3) and 19d (1.14 ± 0.5) vs young at 14d (1.92 ± 0.5) (p≤0.01 A14d vs Y14d for VEGF). However, similar production of VEGF receptor2 was observed. In contrast to young mice, there was significantly increased expression of thrombospondin-2 (TSP-2) in aged mice from 14d (14.6 × 103 ± 7.3 × 103) to 19d (34.9 × 103 ± 17 × 103). We conclude that angiogenesis in aging is not merely delayed, but is altered due to multiple impairments.

A deficit in collagenase activity contributes to impaired migration of aged microvascular endothelial cells

Angiogenesis is impaired in aging. Delayed neovascularization is due, in part, to slowed endothelial cell migration. Migration requires an optimal level of adhesion to matrix proteins, a process mediated by matrix‐degrading metalloproteases (MMPs) such as MMP1. To determine whether impaired angiogenesis in aging is associated with altered synthesis and activity of MMP1, we examined the expression of collagenase and tissue inhibitor of metalloprotease 1 (TIMP1) by immunostain of angiogenic sponge implants from young and aged mice. To characterize the relevance of MMP activity during the movement of aged endothelial cells, the secretion of MMP1 and TIMP1 by late‐passage human microvascular endothelial cells (hmEC aged in vitro) and their non‐aged (young) counterparts was quantified. The migration of aged human microvascular endothelial cells and the effect of inhibition of TIMP1 on the migration of aged hmEC or collagen I was also measured. Relative to young mice, granulation tissue from aged mice showed less expression of collagenase and increased expression of TIMP1. In vitro, aged hmEC were deficient in MMP1 secretion (55 ± 13% relative to young cells) and activity but showed increased expression of TIMP1 (280 ± 109% relative to young cells). Aged hmEC migrated significantly less distance than did young hmEC over a 5‐day period (59 ± 8% relative to young cells). In the presence of a blocking antibody to TIMP1, aged hmEC showed a significant increase in the distance migrated on collagen I over a 5 day period (142 ± 11% relative to untreated aged hmEC). We propose that deficient MMP1 activity contributes to impaired cellular movement in aged microvascular endothelial cells and that perturbations that enhance collagenase activity increase their migratory ability and angiogenic potential. J. Cell. Biochem. 77:116–126, 2000.

Aging and angiogenesis.

Angiogenesis is impaired in aged tissues. It is probable that this deficit contributes to the increased severity of vascular diseases observed in older persons. The changes in angiogenesis that occur with aging have been noted at the molecular, cellular, and physiologic levels of regulation. Components of the neovascular process that are influenced by age include endothelial cells, the hemostatic cascade, neuro-chemical mediators, and growth factors and their cognate receptors. The structural and regulatory components of the matrix scaffold that surround newly formed vessels is also altered in aged tissues. These myriad changes result in delayed and impaired neovascularization. The clinical consequences of the decreased potential of aged tissues to form new vessels is detrimental during the revascularization of the ischemic heart and during the repair of injured tissues, but may be of benefit in slowing the growth of tumors. In this context, clinical strategies to improve the function of the aging vasculature in general, and the angiogenic response in particular, must be targeted to specific disease states in order to maximize the potential benefit to older individuals.

Enhanced cell proliferation and biosynthesis mediate improved wound repair in refed, caloric-restricted mice

Aged mice that have undergone long-term caloric-restriction (CR) have improved health and enhanced longevity in comparison to aged mice that are ad libitum-fed (AL). However, caloric-restriction does not benefit the impaired wound healing of aged mice. To test the hypothesis that CR mice have the capacity for enhanced wound repair, but require a short-term period of additional nutrient intake to show this advantage, we assessed wound healing in CR mice that had been refed (RF) an ad libitum diet for 4 weeks prior to wounding. Two strains of AL young (Y AL) (4-6 months), AL middle-aged (M AL) (15-17 months), and three different, matched cohorts of old mice (O) (30-33 months): O AL, O CR, and O RF were studied. Two full-thickness 4 mm diameter punch biopsy skin wounds were created on the dorsum of each mouse. Animals were sacrificed and wounds were harvested at 1,2,3,5, and 7 days post-wounding. Repair of wounds was slower in O AL and O CR mice compared to Y AL and M AL animals. In contrast, the O RF mice healed similarly to that of the Y AL and M AL mice, as assessed by measures of wound area and histologic criteria. O RF mice demonstrated enhanced synthesis of type I collagen mRNA in comparison to O AL and O CR mice. A greater number of endothelial cells and fibroblasts at the wound edge of the O RF mice exhibited replication in vivo as measured by uptake of BrdU. O RF mice had higher levels of insulin-like binding protein 3 (IGFBP-3). Furthermore, fibroblasts derived from the explant of the punch biopsy of O CR mouse skin revealed enhanced proliferation and contraction in vitro, in comparison to fibroblasts from the O AL mice. In conclusion, O RF mice demonstrate an enhanced capacity to undergo wound repair in comparison to O AL mice. This effect appears to be mediated, in part, by enhanced cell proliferation, contraction, and collagen biosynthesis. In addition, short-term refeeding induced an increase in the serum level of IGFBP-3, the major binding protein for IGF-1. These data confirm that cells from O CR animals have a preserved proliferative, biosynthetic, and contractile capacity, but that an adequate source of nutrients is necessary to demonstrate this advantage in wound healing.

The Effects of Aging on the Molecular and Cellular Composition of the Prostate Microenvironment

Background Advancing age is associated with substantial increases in the incidence rates of common diseases affecting the prostate gland including benign prostatic hyperplasia (BPH) and prostate carcinoma. The prostate is comprised of a functional secretory epithelium, a basal epithelium, and a supporting stroma comprised of structural elements, and a spectrum of cell types that includes smooth muscle cells, fibroblasts, and inflammatory cells. As reciprocal interactions between epithelium and stromal constituents are essential for normal organogenesis and serve to maintain normal functions, discordance within the stroma could permit or promote disease processes. In this study we sought to identify aging-associated alterations in the mouse prostate microenvironment that could influence pathology. Methodology/Principal Findings We quantitated transcript levels in microdissected glandular-adjacent stroma from young (age 4 months) and old (age 20–24 months) C57BL/6 mice, and identified a significant change in the expression of 1259 genes (p<0.05). These included increases in transcripts encoding proteins associated with inflammation (e.g., Ccl8, Ccl12), genotoxic/oxidative stress (e.g., Apod, Serpinb5) and other paracrine-acting effects (e.g., Cyr61). The expression of several collagen genes (e.g., Col1a1 and Col3a1) exhibited age-associated declines. By histology, immunofluorescence, and electron microscopy we determined that the collagen matrix is abundant and disorganized, smooth muscle cell orientation is disordered, and inflammatory infiltrates are significantly increased, and are comprised of macrophages, T cells and, to a lesser extent, B cells. Conclusion/Significance These findings demonstrate that during normal aging the prostate stroma exhibits phenotypic and molecular characteristics plausibly contributing to the striking age associated pathologies affecting the prostate.

Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit

BackgroundDisruption of the blood-brain barrier (BBB) occurs in many diseases and is often mediated by inflammatory and neuroimmune mechanisms. Inflammation is well established as a cause of BBB disruption, but many mechanistic questions remain.MethodsWe used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in mice. BBB disruption was measured using 14C-sucrose and radioactively labeled albumin. Brain cytokine responses were measured using multiplex technology and dependence on cyclooxygenase (COX) and oxidative stress determined by treatments with indomethacin and N-acetylcysteine. Astrocyte and microglia/macrophage responses were measured using brain immunohistochemistry. In vitro studies used Transwell cultures of primary brain endothelial cells co- or tri-cultured with astrocytes and pericytes to measure effects of LPS on transendothelial electrical resistance (TEER), cellular distribution of tight junction proteins, and permeability to 14C-sucrose and radioactive albumin.ResultsIn comparison to LPS-induced weight loss, the BBB was relatively resistant to LPS-induced disruption. Disruption occurred only with the highest dose of LPS and was most evident in the frontal cortex, thalamus, pons-medulla, and cerebellum with no disruption in the hypothalamus. The in vitro and in vivo patterns of LPS-induced disruption as measured with 14C-sucrose, radioactive albumin, and TEER suggested involvement of both paracellular and transcytotic pathways. Disruption as measured with albumin and 14C-sucrose, but not TEER, was blocked by indomethacin. N-acetylcysteine did not affect disruption. In vivo, the measures of neuroinflammation induced by LPS were mainly not reversed by indomethacin. In vitro, the effects on LPS and indomethacin were not altered when brain endothelial cells (BECs) were cultured with astrocytes or pericytes.ConclusionsThe BBB is relatively resistant to LPS-induced disruption with some brain regions more vulnerable than others. LPS-induced disruption appears is to be dependent on COX but not on oxidative stress. Based on in vivo and in vitro measures of neuroinflammation, it appears that astrocytes, microglia/macrophages, and pericytes play little role in the LPS-mediated disruption of the BBB.

Compromised Production of Extracellular Matrix in Mice Lacking Secreted Protein, Acidic and Rich in Cysteine (SPARC) Leads to a Reduced Foreign Body Reaction to Implanted Biomaterials

SPARC (secreted protein, acidic and rich in cysteine), a matricellular glycoprotein, modulates the interaction of cells with the extracellular matrix (ECM). Recently, accelerated cutaneous wound closure and altered deposition of collagen were reported in SPARC-null mice. Herein we asked whether SPARC might influence the foreign body reaction to biomaterial implants. Polydimethylsiloxane (silicone rubber) disks and cellulose Millipore filters were implanted into wild-type and SPARC-null mice. In wild-type animals, significant levels of SPARC were observed in the cells and the ECM comprising the capsules around the implants. After 4 weeks, SPARC-null mice exhibited a significant decrease in the thickness of the foreign body capsule, as compared to that observed in wild-type mice. A significant reduction in capsular vascular density was also associated with the silicone implants in the SPARC-null animals. Electron microscopy revealed that collagen fibers in the capsules produced by SPARC-null mice were smaller and more uniform in size than those in wild-type animals. Furthermore, staining with picrosirius-red showed that the collagen fibers were less mature in SPARC-null than in wild-type mice. The altered ECM resulting in decreased capsular thickness, indicative of an altered foreign body reaction in SPARC-null mice, implicates SPARC as an important modulator of the encapsulation of implanted biomaterials.