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In spring 2022, pink salmon Oncorhynchus gorbuscha smolts were recorded in the UK. Fish were caught in the Rivers Thurso and Oykel in Scotland between 13 and 17 March. To our knowledge, this is the first observation of O. gorbuscha smolts in Europe outside the Scandinavian and Kola peninsulas including other tributaries of the White and Barents Seas. It also provides evidence of successful spawning in 2021, completion of the freshwater phase of the life cycle, and indicates the possibility for potential establishment of an O. gorbuscha population in Great Britain. This article is protected by copyright. All rights reserved.

Cutaneous wound healing is an intricate orchestration of three overlapping phases of repair that encompass numerous cell types, signalling cascades, and microenvironment modifications to reach a successful resolution. Disruption of any of these steps will create an abnormal healing response resulting in either ulceration or excessive scarring. It has become evident that the extracellular matrix and its associated components are key orchestrators during this process. One of these essential matrix proteins is decorin, a small leucine-rich proteoglycan (SLRP) that acts as a regulator of collagen fibrillogenesis and a non-competitive inhibitor of multiple growth factors signalling cascades. Decorin is a necessary shut-off switch for the pro-reparative mechanism of the tissue replacement phase and limits the occurrence of hypertrophic scarring by preventing excessive repair. We investigated the use of decorin as a therapeutic by administering the matrix protein anchored in a slow-release coacervate in a hypertrophic scarring mouse model. The results show that early wound healing phase measurements exhibit little difference in performance compared to our coacervate-only baseline or HB-EGF-treated control mice. However, during the resolution phase of wound healing, the decorin-treatment significantly reduces cutaneous thickness, enhances collagen alignment, and improves overall wound scoring in the mice. Thus, mice treated with decorin display better healing outcomes and could limit the hypertrophic scarring phenotype in the coacervate only, and HB-EGF controls. These results suggest that decorin may be a promising tool and alternative therapy for patients who suffer from over-exuberant matrix deposition during wound healing.

SARS-CoV-2 triggers a dysregulated innate immune system activation. As the mevalonate pathway (MVP) prevents the activation of inflammasomes and cytokine release and regulates endosomal transport, compromised signaling could be associated with the pathobiology of COVID-19. Prior transcriptomic studies of host cells in response to SARS-CoV-2 infection have not reported to date the effects of SARS-CoV-2 on the MVP. In this study, we accessed public data sets to report in silico investigations into gene expression. In addition, we proposed candidate genes that are thought to have a direct association with the pathogenesis of COVID-19, and which may be dependent on signals derived from the MVP. Our results revealed dysregulation of genes involved in the MVP. These results were not found when investigating the gene expression data from host cells infected with H3N2 influenza virus, H1N1 influenza virus, or respiratory syncytial virus. Our manually curated gene set showed significant gene expression variability in A549 cells infected with SARS-CoV-2, as per Blanco-Melo et al. data set (GSE147507). In light of the present findings, SARS-CoV-2 could hijack the MVP, leading to hyperinflammatory responses. Prompt reconstitution of this pathway with available agents should be considered in future studies.

BACKGROUND: Antidepressant use later in pregnancy has been associated with preeclampsia and postpartum hemorrhage (PPH) in some studies. OBJECTIVE: To evaluate the association between patterns of prenatal antidepressant dose across gestation and risk of precclampsia and PPH. METHODS: We utilized OptumLabs® Data Warehouse (2012–2016) administrative health care claims, identifying 226,932 singleton live-born deliveries for this retrospective cohort study. Antidepressant dispensing doses were converted to fluoxetine equivalents. Using k-means longitudinal, we identified women with similar patterns of antidepressant exposure, i.e., trajectory groups, during the first 20 and 35 gestational weeks. We estimated risk ratios (RR) and 95% confidence intervals (CI) for the association between trajectory groups and preeclampisa (20-week groups) and PPH (35-week groups), adjusting for demographics, comorbidities, and other psychotropic medications. Linear trend tests assessing increasing risk of the outcomes across groups were performed. RESULTS: Among 15,041 (6.6%) pregnancies exposed to an antidepressant, the following trajectory groups were identified: A-low exposure, starting pregnancy at ~10mg/day, with 1(st) trimester reduction/discontinuation, B-low sustained exposure of ~20 mg/day, C-moderate exposure (~40mg/day) with 1(st) trimester reduction/discontinuation, D-moderate sustained exposure of ~40 mg/day, and E-high sustained exposure of ~75mg/day. In the low exposure with reduction/discontinuation trajectory, risks were 8.2% for preeclampsia and 2.7% for PPH. Compared with this group, low, moderate, and high sustained trajectories were associated with preeclampsia [adjusted (a)RR: 1.17 (95% CI: 1.01, 1.34), aRR: 1.31 (95% CI: 1.12, 1.54), aRR: 1.41 (95% CI: 1.05, 1.90), respectively] and PPH [aRR: 1.32 (1.05, 1.66), aRR: 1.35 (95% CI: 1.03,1.78), aRR: 2.51 (95% CI: 1.69, 3.71), respectively]; p

No abstract available.

BACKGROUND AND METHODS Prostate cancer frequently expresses an osteomimetic phenotype, but it is unclear how it is regulated and what biological and clinical implications it confers. Because mechanical forces physiologically regulate bone-remodeling activity in osteocytes, we hypothesized that mechanical action of fluid flow (MAFF) at the cancer microenvironment may similarly foster prostate cancer cell osteomimicry. RESULTS We showed that in vitro MAFF on androgen-dependent (LNCap) and androgen-independent (PC3) prostate cancer cells remarkably increased OPG, VEGF, RunX2, PTH1R, and PTHrP gene expression in both cell lines irrespective of their androgen dependency. MAFF also altered the cytokine secretion pattern of prostate cancer cells, including Ang2, SCF, and TNFα increase with TRAIL decrease in the supernatant of both cell lines; preferential increase of Leptin and PDGF-BB in LnCap and of VEGF, IL-8, and G-CSF in PC3; and exclusive increase of FGFβ, MIF, and PECAM-1 with HGF decrease in LnCap, and of TGBβ1, HGF, M-CSF, CXCL1, and CCL7 with NGF decrease in PC3. Murine MLO-Y4 osteocyte-conditioned medium (CM) abrogated M-CSF, G-CSG, IL-8, TNFα, and FGFβ secretion-stimulating activity of mechanical stimulation on PC3 cells, and did the opposite effect on LnCap cells. However, MAFF fostered osteomimetic gene expression response of PC3 cells, but not of LnCap cells, to mechanically stimulated osteocyte-CM. Moreover, it abrogated TNFα and IL-8 secretion inhibitory effect of osteocyte-CM on mechanically stimulated PC3 cells and G-CSF, TNFα, and FGFβ-stimulating effect on mechanically stimulated LnCap cells. CONCLUSIONS MAFF activated osteoblast-like phenotype of prostate cancer cells and altered their responses to osteocyte soluble factors. It also induced osteocyte production of osteomimetic gene expression- and cytokine secretion-stimulating factors for prostate cancer cells, particularly, when they were mechanically stimulated. Importantly, MAFF induced a prometastatic response in androgen-independent prostate cancer cells, suggesting the interest of mechanical stimulation-dependent transcription and secretion patterns as diagnostic biomarkers, and as therapeutic targets for the screening of bone-metastasizing phenotype inhibitors upregulated during prostate cancer cell response to MAFF at the cancer microenvironment. Prostate 77:321–333, 2017. © 2016 Wiley Periodicals, Inc.

Pericytes have generally been considered in the context of stabilizing vessels, ensuring the blood barriers, and regulating the flow through capillaries. However, new reports suggest that pericytes may function at critical times to either drive healing with minimal scarring or, perversely, contribute to fibrosis and ongoing scar formation. Beneficially, pericytes probably drive much of the vascular involution that occurs during the transition from the regenerative to the resolution phases of healing. Pathologically, pericytes can assume a fibrotic phenotype and promote scarring. This perspective will discuss pericyte involvement in wound repair and the relationship pericytes form with the parenchymal cells of the skin. We will further evaluate the role pericytes may have in disease progression in relation to chronic wounds and fibrosis.

Pericytes are mural cell that have been found to play important roles in promoting blood vessel development and regulating blood flow. The signals that attract pericytes to maturing vessels during the resolution phase of wound healing are unknown. In this study, we examine the role of the chemokine receptor CXC receptor 3 (CXCR3) ligands, as they are produced by maturing endothelial cells. Pericytes isolated from muscle and retina were found to by and large only express the B-isoform of CXCR3 (CXCR3B), with expression being independent of the mitotic state of the cells. Pericyte stimulation with the CXCR3 ligands Mig (CXCL9), IP-9/I-TAC (CXCL11), or IP-10 (CXCL10) resulted in the activation of ERK but not AKT. Treatment with Mig or IP-9, but not IP-10, enhanced p38(MAPK) phosphorylation. Interestingly, while cyclic adenosine monophosphate is generated downstream of CXCR3B in other cells, protein kinase A activation was not observed in these pericytes when treated with these three CXCR3 ligands. The increase in ERK activity resulted in a slight increase in cell transmigration, with the inhibition of ERK leading to a decrease in CXCR3B mediated migration and inhibition of p38(MAPK) reducing transmigration through small pores. These ligands did not affect proliferation. These data are the first to characterize CXCR3B as the predominant isoform expressed on pericytes, and was found on these diverse cells isolated from both muscle and eye. We also show that CXCR3B signaling stimulates transmigration of barrier pores in pericytes as opposed to its inhibitory affects on endothelial cells and fibroblasts. These findings characterize a novel role for the CXCR3B in regulating cellular function. Taken together these data show a role for CXCR3B in regulating pericyte function.

The adverse physiological and psychological effects of scars formation after healing of wounds are broad and a major medical problem for patients. In utero, fetal wounds heal in a regenerative manner, though the mechanisms are unknown. Differences in fetal scarless regeneration and adult repair can provide key insight into reduction of scarring therapy. Understanding the cellular and extracellular matrix alterations in excessive adult scarring in comparison to fetal scarless healing may have important implications. Herein, we propose that matrix can be controlled via cellular therapy to resemble a fetal-like matrix that will result in reduced scarring.

Objectives: Vocal fold injury can be irreversible, leading to vocal fold scarring, with permanent functional effects and no optimal treatment. A porcine model of vocal fold scarring was used to test effects of decorin and primed vocal fold fibroblasts in vitro using a cell migration assay and immunoblotting, and by using functional measurements of porcine larynges and excised porcine vocal folds. Methods: In vitro: primary pig vocal fold fibroblasts (PVFFs) were subjected to cell migration assays (scratch) and treated with decorin 20 μg/mL, hepatocyte growth factor (HGF) 200 ng/mL, epidermal growth factor (EGF) 1 nM, or transforming growth factor-β1 10 ng/mL. Cells also underwent decorin dose response testing. Scratch assays were analyzed in MetaMorph® Imaging; cell lysates were processed for MMP-8 and type I collagen content. Eleven pigs underwent unilateral vocal fold stripping procedures. At day 3 postoperatively, subjects underwent superficial injection into the affected vocal fold either with decorin 20 μg/mL or 1 × 106 HGF-primed fibroblasts. Larynges were harvested and either used for ex vivo laryngeal testing or for rheological testing. Results: Scratch assay indicated significantly reduced cell migration in PVFFs treated with decorin or HGF. MMP-8 production was increased (P

Disruption of the actin cytoskeleton is a feature of malignant cells that correlates with dysregulated expression of various actin-binding proteins (ABPs) (Wang et al., 1996; Clark et al., 2000). The malignant phenotype of tumor cells often can be reversed by experimental restoration of ABP expression, suggesting misregulation of ABPs could contribute directly to malignancy (Tanaka et al., 1995; Nikolopoulos et al., 2000). Profilin-1 (Pfn1), a ubiquitously expressed G-actin binding protein, is significantly downregulated in various types of adenocarcinoma (breast, pancreatic, hepatic) (Janke et al., 2000; Gronborg et al., 2006; Wu et al., 2006). This raises a fundamental question as to whether loss of Pfn1 expression contributes to malignant progression of tumor cells. We found that upon downregulation of Pfn1, normal human mammary epithelial cells (HMEC) exhibit disruption of cell–cell adhesion (a hallmark change that facilitates epithelial cell dissemination and migration during cancer progression) (Zou et al., 2007). Furthermore, silencing Pfn1 expression leads to increased motility and invasiveness of breast cancer cell lines and conversely, overexpression of Pfn1 dramatically suppresses the aggressive phenotype of breast cancer cells (Roy and Jacobson, 2004; Zou et al., 2007). These findings collectively suggest that Pfn1 downregulation may contribute to breast cancer invasion and metastasis. Although Pfn1 was initially identified as a G-actin sequestering protein (Karlsson et al., 1977), it can promote actin polymerization by acting as an efficient ADP-to-ATP exchanger on G-actin and adding ATP-G-actin to the barbed but not pointed ends of actin filaments (Witke, 2004). Consistent with this model, Pfn1 depletion induces a significant reduction in F-actin content in various cell types (Ding et al., 2006; Zou et al., 2007). Gene deletion of both Pfn1 and Pfn2 [another variant of Pfn1 that is mainly expressed in the nervous system in vertebrates (Lambrechts et al., 2000)] caused impaired motility of Dictyostelium amebae, providing direct evidence for Pfn’s involvement in migration of eukaryotic cells (Haugwitz et al., 1994). Further support for Pfn1’s requirement in cell migration came from studies that showed cell motility defects in a chickadee (Pfn1 homolog)-null Drososphila mutant (Verheyen and Cooley, 1994) and in human vascular endothelial cells in which Pfn1 expression was silenced (Ding et al., 2006). Several lines of experimental evidence including slower actin-driven intracellular propulsion of bacterial pathogens (Loisel et al., 1999; Mimuro et al., 2000), impaired membrane protrusion of vascular endothelial cells as a consequence of Pfn1 depletion (Ding et al., 2006), and very recently, induction of lamellipodia by Pfn1 in a growth-factor insensitive mechanism (Syriani et al., 2008) all point to a key role of Pfn1 in driving lamellipodial protrusion during cell migration. Several important classes of ABPs that either promote nucleation and/ or elongation of actin filaments at the leading edge including those belonging to Ena/VASP [Enabled/vasodilator stimulated phosphoprotein (Ferron et al., 2007)], Wiskott–Aldrich syndrome protein [WASP; example: Neuronal or N-WASP (Suetsugu et al., 1998), WAVE or WASP-associated verprolin homology (Miki et al., 1998)] family are physiological ligands that bind Pfn1 via polyproline-rich motifs. It has thus been proposed that these proline-rich proteins, when activated by signals, act as scaffolds to spatially recruit Pfn1-actin complex to the sites of actin assembly during cell migration (Holt and Koffer, 2001). In addition to actin and polyproline ligands, Pfn1 also binds to membrane phosphoinositides (phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]), phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3] (Lu et al., 1996). Although the physiological implications of Pfn1’s binding to membrane lipids have not been explored in detail, based on Pfn1’s ability to inhibit PI(4,5)P2 hydrolysis it has been proposed that Pfn1 might also regulate phosphoinositide metabolism in cells (Goldschmidt-Clermont et al., 1990). In fact, a recent study by our group has demonstrated that overexpression of Pfn1 in breast cancer cells dramatically suppresses growth-factor induced PI(3,4,5)P3 generation (Das et al., 2009), Since membrane phosphoinositides are critical regulators of actin cytoskeleton, Pfn1 could be an important nexus between phosphoinositide-derived signaling and cell motility. Given the plethora of evidence demonstrating importance of Pfn1 in driving actin polymerization at the leading edge and hence playing a critical role in cell migration, it is not clear how reduced Pfn1 expression might contribute to faster motility of breast cancer cells. This was the overall goal of the present study where we have now linked loss of Pfn1 expression and hyper-motile phenotype of breast cancer cells through a Ena/VASP-dependent mechanism.

Wound healing is a complex, orchestrated series of biological events that is controlled by extracellular components that communicate between cell types to re-establish lost tissue. We have found that signaling by ELR-negative CXC chemokines through their common CXCR3 receptor is critical for dermal maturation during the resolving phase. In addition there needs to be complete maturation of the epidermis and regeneration of a delineating basement membrane for proper functioning. The role of this ligand-receptor system appears confounding as one ligand, CXCL4/(PF4), is present during the initial dissolution and two others, CXCL10/(IP-10) and CXCL11/(IP-9/I-TAC), are expressed by keratinocytes in the later regenerative and resolving phases during which the basement membrane is re-established. We examined CXCR3 signaling role in healing using a mouse lacking this receptor, as all three ligands act solely via the common receptor. Reepithelialization was delayed in CXCR3-deficient mice in both full and partial-thickness excisional wounds. Even at 90 days postwounding, the epidermis of these mice appeared less mature with lower levels of E-cadherin and cytokeratin 18. The underlying basement membrane, a product of both dermal fibroblasts and epidermal keratinocytes, was not fully established with persistent diffuse expression of the matrix components laminin 5, collagen IV, and collagen VII throughout the wound bed. These results suggest that CXCR3 and its ligands play an important role in the re-establishment of the basement membrane and epidermis. These studies further establish the emerging signaling network that involves the CXCR3 chemokine receptor and its ligands as a key regulator of wound repair.

A technique using 51 chromium-labelled erythrocytes was used to measure blood volume in Scyliorhinus canicula following long-term and acute salinity transfers. Basal whole-blood volume was 5·6 ± 0·2 ml 100 g−1 (mean ±s.e.), this increased (6·3 ± 0·2 ml 100 g−1) following +14 day acclimation to 80% sea water (SW) and decreased (4·6 ± 0·2 ml 100 g−1) following acclimation to 120% SW. These changes were shown to be primarily due to changes in plasma volume, with no significant changes in extrapolated red-cell volume being demonstrated. Blood volume was also measured in the same animals during 10 h acute transfer to 100% SW. Plasma volume in S. canicula during acclimation from 80% SW was significantly reduced (4·5 ± 0·3 ml 100 g−1) after 6 h of transfer to 100% SW. Blood volume in animals during acclimation from 120% SW was significantly increased (4·8 ± 0·2 ml 100 g−1) after 4 h of acute transfer. The osmoregulatory implications of these different timeframes during hyposaline and hypersaline transfer are discussed, along with the importance of this in vivo technique as context for in vitro studies with haemo-dynamic stimuli.

Angiogenesis plays a critical role in wound healing. In wound healing new blood vessels are generated, then later in scar-resolving stages of the repair process, these newly formed blood vessels undergo destabilization and significant regression. ELR-negative chemokines have been shown to have angiostatic properties and are produced in high concentration late in the granulation tissue and resolving stage of wound repair. The receptor for the ELR-negative chemokines, CXCR3, is expressed on endothelial cells but its function is not known. To understand the physiologic role CXCR3 plays in regulating endothelial function, we analyzed the ability of the CXCR3 ligand, IP-10, to inhibit endothelial cell tube formation. IP-10 was able to inhibit endothelial cell tube formation alone and prevent VEGF-induced tube formation on Matrigel®. Treatment of endothelial cells with IP-10 significantly inhibited endothelial motility. Thus, the ELR-negative chemokines inhibit angiogenesis by inhibiting endothelial motility. To identify the signaling pathway(s) mediated by CXCR3 that inhibit endothelial cell motility, we looked at the regulation of key processes. As CXCR3 is a receptor known to generate cAMP, as we show in these cells, we examined PKA-mediated inhibition of calpain. Treatment of endothelial cells with VEGF induced the activation of m-calpain but costimulation with IP-10 significantly decreased m-calpain activity. Using cAMP analogs, 8-Br-cAMP, a PKA activator, is able to inhibit VEGF- induced motility and Rp-8-Br-cAMP, a PKA inhibitor, is able to reverse the inhibitory effects of IP-10 on endothelial migration and calpain activation. To further demonstrate that m-calpain is important in endothelial motility we show that siRNA knockdown of m-calpain inhibited VEGF induced migration. These data indicate that activation of CXCR3 inhibits endothelial migration through the PKA-mediated inhibition of m-calpain. These findings provide new targets to modulate the resolution phase of wound repair.

We find that the ELR-negative CXC chemokines, CXCL10 (IP-10) and CXCL11(IP-9), expressed during wound repair, with IP-9 being produced by redifferentiated keratinocyte. Immunohistochemical analysis for IP-9 on human full thickness wound tissues collected from day 5 confirmed that IP-9 is a wound response protein. We have shown in in vitro coculture systems that IP-9 can serve as an autocrine factor promoting motility in keratinocyte while limiting motility in dermal fibroblasts. These effects occur via modulation of the intracellular proteases calpain isoforms that regulate cell adhesion during motility. Thus, the function of these chemokines in vivo is not obvious. To probe this, we have explored wound repair in mice lacking the receptor for these ligands, CXCR3. Quantitative analysis of IP-9 expression in wounds of these mice collected from day 2 to day 20 showed a significant increase in IP-9 levels in CXCR3-/- mice compared to wild type, which is expected if receptor-mediated feedback attenuation is lost. Full and partial thickness wound healing experiments on these mice showed a significant delay in CXCR3-/- mice when compared to wild type. Histochemical analysis of tissues collected from both full and partial thickness wounds demonstrated differences in epithelialization, granulation tissue formation, and angiogenesis in CXCR3-/- mice when compared to wild type. Collagen, the major extracellular matrix protein organization were analyzed by trichrome and picrosirus red staining methods. We are also examining the effect of this lost receptor on promotility signaling from EGF receptors. These in vivo and in vitro studies confirm the pathophysiologic role of the chemokines IP-9 and IP-10 and their cognate receptor CXCR3 during wound repair.

Select epidermal growth factor (EGF)-like (EGFL) repeats of human tenascin cytotactin (tenascin C) can stimulate EGF receptor (EGFR) signaling, but activation requires micromolar concentrations of soluble EGFL repeats in contrast to subnanomolar concentrations of classical growth factors such as EGF. Using in silico homology modeling techniques, we generated a structure for one such repeat, the 14th EGFL repeat (Ten14). Ten14 assumes a tight EGF-like fold with truncated loops, consistent with circular dichroism studies. We generated bound structures for Ten14 with EGFR using two different approaches, resulting in two distinctly different conformations. Normal mode analysis of both structures indicated that the binding pocket of EGFR exhibits a significantly higher mobility in Ten14–EGFR complex compared to that of the EGF–EGFR complex; we hypothesized this may be attributed to loss of key high-affinity interactions within the Ten14–EGFR complex. We proved the efficacy of our in silico models by in vitro experiments. Surface plasmon resonance measurements yielded equilibrium constant KD of 74 µM for Ten14, approximately three orders of magnitude weaker than that of EGF. In accordance with our predicted bound models, Ten14 in monomeric form does not bind EGFR with sufficient stability so as to induce degradation of receptor, or undergo EGFR-mediated internalization over either the short (20 min) or long (48 h) term. This transient interaction with the receptor on the cell surface is in marked contrast to other EGFR ligands which cause EGFR transit through, and signaling from intracellular locales in addition to cell surface signaling. J. Cell. Physiol. 211: 748–758, 2007. © 2007 Wiley-Liss, Inc.

To investigate hepatocyte growth factor (HGF) primed fibroblasts and decorin application on skin and vocal fold fibroblasts in vitro and in vivo in rabbit vocal fold scar model.Vocal fold and skin fibroblasts underwent five in vitro treatment conditions: control, epidermal growth factor, HGF, both decorin and HGF, and decorin alone. Hyaluronic acid and collagen enzyme-linked immunosorbent assays were performed. In vivo, 12 rabbits underwent unilateral vocal fold stripping. Injured vocal folds were then injected with skin fibroblasts, HGF, HGF-primed fibroblasts and decorin, or decorin. Outcomes included histologic and lamina propria height analyses.In vitro, HGF increased hyaluronic acid synthesis in vocal fold fibroblasts (P0.001). HGF and decorin treatment diminished collagen secretion (P0.01). In vivo, histologic findings indicated minimal difference in collagen amount between treatment groups.HGF and decorin together may decrease collagen production by skin and vocal fold fibroblasts. Fibroblast transplantation into scarred vocal folds has equivocal benefit.

Recently, extracellular matrix components have been shown to contain domains that can interact with and activate receptors with intrinsic tyrosine kinase activity. These receptor tyrosine kinases are strong mediators of the cell responses of proliferation, migration, differentiation, and dedifferentiation. However, an interesting question is raised as to why cells would present growth factor receptor ligands in such a manner, as the majority of growth factors are small, soluble, or only transiently tethered ligands. With the exception of the discoidin domain receptors that bind collagen, the other described domains interact with a receptor that binds ubiquitous soluble peptide growth factors, the epidermal growth factor receptor. Unlike traditional growth factors, these individual "matrikine" domains within tenascin-C, laminin, collagen, and decorin possess relatively low binding affinity (high nanomolar or micromolar) and are often presented in multiple valency. The presentation of ligands within the extracellular matrix in this fashion might allow for unique biochemical and physiological outcomes. This new class of "matrikine" ligand may be critical for wound healing, as the majority of known extracellular matrix components possessing matrikines play a strong role, or are presented uniquely, during skin repair. Tenascin-C expression, for instance, is uniquely regulated spatially and has been proposed to present pro-migratory tracks during skin repair through its epidermal growth factor-like repeats. The epidermal growth factor-like repeats of laminin-5 act as cryptic ligands revealed upon matrix metalloproteinase-2 degradation of the surrounding extracellular matrix. The deletion of the discoidin domain receptors 1 and 2 for collagen have negative consequences on the role of fibroblasts and epithelial cells for matrix metalloproteinase production, migration, proliferation, and extracellular matrix turnover. Finally, decorin can bind to, inhibit, and down-regulate epidermal growth factor receptor levels and signaling, suggesting a tonic role of the epidermal growth factor binding domain of decorin in the resolution of wound healing. We provide a model framework for further studies into this emerging class of signals.

Keloids, which overgrow the boundaries of the original injury, represent aberrations in the fundamental process of wound healing that include over-abundant cell in-migration, cell proliferation, and inflammation, as well as increased extracellular matrix synthesis and defective remodeling. To understand the key events that result in the formation of these abnormal scars would open new avenues for better understanding of excessive repair, and might provide new therapeutic options. We examined epidermal growth factor receptor (EGFR)-induced cell motility in keloid fibroblasts, as this receptor initiates cell migration during normal wound repair. We show that keloid fibroblasts respond to EGF-induced cell migration but the response is somewhat diminished compared to normal adult fibroblasts (approximately 30% reduced); the mitogenic response was similarly blunted (approximately 5% reduced). Keloid fibroblasts express near normal levels of EGFR (82%), but show a much more attenuated activation of EGFR itself and the motility-associated phospholipase C-gamma. This was reflected in part by rapid loss of EGFR upon exposure to EGF. Interestingly, while extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) activation was relatively robust in keloid fibroblasts, the downstream triggering of the motility-associated calpain activity was blunted. This was reflected by high cell-substratum adhesiveness in the keloid fibroblasts. Thus, the blunted migratory response to EGF noted in keloid fibroblasts appears due to limited activation of two important biochemical switches for cell motility.

Combining engineering analyses and mathematical modeling with intervention and detection methodologies at the molecular level will allow manipulation of intracellular signal transduction pathways, and therefore rational control of functional processes central to medicine and biotechnology. We have formulated a simple mathematical model of a key signaling pathway required for regulated migration of fibroblasts and other cell types: activation of the intracellular enzyme phospholipase C (PLC) mediated by epidermal growth factor receptor (EGFR) and a multitude of other transmembrane receptors. One of the interesting features of this pathway is that the substrate of PLC, the lipid phosphatidylinositol (4,5)-bisphosphate (PIP(2)), is turned over quite rapidly and must be constantly resupplied to the plasma membrane by a known transfer mechanism. The model, which accounts for regulation of PIP(2) concentration, is sufficiently detailed to explain unique quantitative features of recent experimental data. We find that competitive pathways that deplete PIP(2) from the membrane, as well as receptor-mediated enhancement of PIP(2) supply, must be significant for agreement between model and experiment. Importantly, the mechanistic nature of the model also allowed us to predict the efficacy of various molecular intervention strategies, including overexpression of wild-type and variant proteins in the pathway as well as treatment with specific drug inhibitors. For many parameter conditions the intuitive strategy of targeting the enzyme itself is actually predicted to be relatively inefficient, with a novel and potentially useful alternative being disruption of the reactant supply mechanism.

For effective migration, cells must establish an asymmetry in cell/substratum biophysical interactions permitting cellular protrusive and contractile motive forces to produce net cell body translocation; often this is superficially manifested as a polarized cell shape. This change is most easily noted for epithelial cells, which typically undergo a mesenchymal transition prior to rapid motility, and for hematopoietic cells, which must transition from non-adherent to adherent states. These two situations entail dramatic changes that also involve cell-cell contact and differentiation-related changes, and thus introduce confounding events and signals in defining control elements. Hence, a simpler biochemical and biophysical model system may be useful for gaining fundamental insights into the underlying mechanisms. Fortunately, even relatively "uniform" fibroblasts also undergo an initial shape change to commence locomotion. Investigators have recently begun to probe underlying signals that contribute to the reorganization of the actin cytoskeleton. We describe here a model for fibroblast shape changes involved in epidermal growth factor (EGF) stimulation of motility, focusing on signals through EGF receptor (EGFR) -mediated pathways influencing cytoskeletal organization and cell/substratum adhesion. We present new data addressing specifically phospholipase C-gamma (PLCgamma) pathway activation of actin-modifying proteins, including gelsolin, that contributes to these changes and promotes cell migration by increasing the fraction of cells in a motility-permissive morphology and the time spent in such a state.

BACKGROUND Dolastatin 10 is an anticancer peptide isolated from the sea hare, Dolabela auricularia, which is currently in phase I trials. METHODS The effects of dolastatin 10 on the DU-145 human prostate cancer cell line were studied both in tissue culture and in athymic nude mice. In tissue culture, after dolastatin 10 treatment, cell cycle kinetics were measured using propidum iodide, apoptosis was estimated using the TUNEL assay, and tubulin architecture studied by direct immunofluorescence. RESULTS At concentrations of 1 nM (IC50 = 0.5 nM), dolastatin 10 completely inhibited the growth in tissue culture of human prostate cancer DU-145 cells. Growth inhibition was correlated with the arrest of these cells in G2/M and alpha-tubulin depolymerization. In athymic mice at a dose of 5 micrograms every 4 days i.p., dolastatin 10 blocked the diaphragmatic invasion of DU-145 tumor cells. CONCLUSIONS Dolastatin 10 is a novel marine-derived compound with activity in the treatment of human prostate cancer in animals. The mechanism of action of this agent involves tubulin depolymerization but not the induction of apoptosis.

The effects of arginine vasotocin on the pattern of glomerular perfusion in an elasmobranch fish were examined using an in situ perfused renal trunk preparation. A significant antidiuresis was coupled with a marked reduction in the proportion of filtering glomeruli (86%-25%) and an increase in the proportion of perfused but not filtering (10%-53%) and non-perfused glomeruli (4%-22%). However, the reduction in filtering glomeruli was greater than predicted by measurements of urine flow rate and glomerular filtration rate alone, suggesting that alterations in single nephron glomerular filtration rate may occur.

Peptide growth factors and other receptor-binding cytokine ligands are of interest in contemporary molecular health care approaches in applications such as wound healing, tissue regeneration, and gene therapy. Development of effective technologies based on operation of these regulatory molecules requires an ability to deliver the ligands to target cells in a reliable and wellcharacterizable manner. Quantitative information concerning the fate of peptide ligands within tissues is necessary for adequate interpretation of experimental observations at the tissue level and for truly rational engineering design of ligand-based therapies. To address this need, we are undertaking efforts to elucidate effects of key molecular and cellular parameters on temporal and spatial distribution of cytokines in cell population and cell/matrix systems. In this article we summarize some of our recent findings on dynamics of growth factor depletion by cellular endocytic trafficking, growth factor transport through cellular matrices, and growth factor production and release by autocrine cell systems.

Acute Chest Syndrome (ACS) continues to be a major source of morbidity and mortality among patients with sickle cell disease. It is characterized by the presence of pleuritic chest pain, fever, raises on lung auscultation, and pulmonary infiltrates on chest x-ray [Castro et all: Blood 84:643-649]. The pathophysiology of this disorder remains poorly understood leading to the descriptive term "Acute Chest Syndrome" designated by Charache et al. [Arch Intern Med 139:67-69, 1979]. Typical bacterial pathogens are seldom isolated in adults, although they play a significant role in the pathogenesis of this entity in children. Until recently, the technology to accurately study viral infection as a precipitating cause of ACS has been unavailable. Parvovirus B19 is being increasingly recognized as an important human pathogen, and has been established as the cause of transient "aplastic crisis" in patients with sickle cell diseases [Saarien et al: Blood 67:-11411-11417, 1986; Young: Sem Hematol 25:159-172, 1988]. We present three patients with hemoglobin SC variant of sickle cell disease who developed ACS in association with acute parvovirus B19 infection, one of which died of respiratory failure. Parvovirus B19 infection was established by polymerase chain reaction for parvovirus B19 DNA, and the presence of parvovirus B19 specific IgM antibodies. These cases suggest that parvovirus B19 may be associated with more than self-limited illness in patients with sickle cell disease, and that this ubiquitous virus may merit further study as a precipitating cause of ACS.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) elicit quantitatively different cell proliferation responses even though they act via a common receptor, the epidermal growth factor receptor (EGFR). We hypothesized that differential cellular trafficking of available ligand is responsible for the different mitogenic responses elicited by EGF and TGF alpha. Mitogenesis and ligand depletion were determined simultaneously in NR6 mouse fibroblasts expressing either wild-type (WT) or internalization-deficient cytoplasmic domain-truncated (c'973) EGFR. Thus we could determine the effects of both ligand-induced and low level constitutive ligand/receptor processing. For a given initial amount of growth factor, TGF alpha is a weaker stimulus than EGF in cells expressing either form of the EGFR. This difference in the mitogenic potencies correlates with increased depletion of TGF alpha observed during the growth assays. When this difference in ligand depletion is accounted for, or minimized, EGF and TGF alpha elicit quantitatively similar growth responses. Therefore, the relative mitogenic potencies of EGF and TGF alpha depend on ligand availability, as determined by the cellular trafficking of these ligands in conjunction with environmental circumstances. Interestingly, our data demonstrate that TGF alpha can be a less potent mitogenic stimulus than EGF under conditions where ligand availability is limited. Further, in our assays, differences in ligand processing are sufficient to explain the different mitogenic potencies of these growth factors in either of the receptor trafficking scenarios. Our results suggest a model of regulation of hormone responsiveness which favors dissociative ligands (such as TGF alpha) in receptor-limited situations and non-dissociative ligands (such as EGF) in the face of high receptor levels.

We describe experiments comparing the proliferation responses to epidermal growth factor (EGF) by NR6 fibroblasts expressing genetically engineered epidermal growth factor receptors (EGFRs). These cells present either wild-type (WT) EGFR or a cytoplasmic domain-truncated (c′973) EGFR that exhibits a decreased ligand-induced internalization rate constant. In two distinct in vitro proliferation assays, with or without medium replenishment, we measured the specific cell proliferation rate constants and EGF depletion kinetics for both WT and c′973 cells. When EGF depletion is minimized by replenishment, the EGF concentration dependencies of the two cell types are similar, whereas when EGF depletion is not prevented, maximal proliferation of WT cells requires an initial EGF concentration that is approximately 10× that required by c′973 cells. However, when EGF depletion is accounted for, the dependencies of growth rate for the two cell types on the current EGF concentration in both assays are essentially identical. Our results demonstrate that diminished depletion of EGF from the extracellular medium is a major reason for increased mitogenic sensitivity to EGF by cells possessing internalization-deficient receptors.

Keloids are scars that overgrow the original boundaries of the injury, involving aberrant functioning of the fibroblasts. We have found that during normal wound healing the ELR-negative CXC chemokines IP-9 (CXCL11) and IP-10 (CXCL10) act to limit fibroblast immigration. Our initial goal was to determine if keloid fibroblasts failed to respond to this ‘stop’ signal. Immunohistochemical and immunocytochemical studies on keloid fibroblasts show that these lesions express excessive amounts of IP-9 and CXCR3 receptor. The protein levels of CXCR3 receptor was also not dissimilar between normal and keloid fibroblasts. Though, the expression of the protein and the receptor seems to be normal, the fibroblasts derived from these abnormal lesions did not respond to IP-9 during EGF induced cell migration. We have extended these findings to determine whether other genes are differentially expressed by keloid fibroblasts. The immediate first gene that interest us was the mRNA levels of CXCR3 receptor (GPR9) wherein, we did not find any significant difference between normal and keloid fibroblasts. Further, we also found interestingly, that genes related to tumor progression are up regulated in keloid fibroblasts than the normal fibroblasts. The marker genes like calgranulin, BCL-2 associated anthogen 4 (BAG4), and dual specificity phosphatase 1 (DUSP1). Certain transcription factors like GATA-6 and SpiB are also increased. Apart, the classical growth factor and growth factor receptor related genes which includes, bone morphogenetic protein BMP-1, heparin binding EGF like growth factor, EGF like repeats discoidin1, IGFBP-5 and 3, EphB1&B2, tumor necrosis factor alpha induced protein, protein tyrosine phosphatase receptor, serine threonine kinase 6 along with chondroitin sulfate proteoglycan brevican and versican and vitronectin are also up regulated. The newl identified genes will open new avenues in targeting keloid lesions by understanding the basic signaling mechanism of these specific proteins. This work was supported by grants from NIH/NIGMS and PPRTP.