Thymosin ß4 Promotes Dermal Healing.

No agent has been identified that significantly accelerates the repair of chronic dermal wounds in humans. Thymosin beta 4 (Tß4) is a small, abundant, naturally occurring regenerative protein that is found in body fluids and inside cells. It was found to have angiogenic and antiinflammatory activity and to be high in platelets that aggregate at the wound site. Thus we used Tß4 initially in dermal healing. It has since been shown to have many activities important in tissue protection, repair, and regeneration. Tß4 increases the rate of dermal healing in various preclinical animal models, including diabetic and aged animals, and is active for burns as well. Tß4 also accelerated the rate of repair in phase 2 trials with patients having pressure ulcers, stasis ulcers, and epidermolysis bullosa wounds. It is safe and well tolerated and will likely have additional uses in the skin and in injured organs for tissue repair and regeneration.

Thymosin Beta 4: A Potential Novel Therapy for Neurotrophic Keratopathy, Dry Eye, and Ocular Surface Diseases.

Chronic ocular surface diseases such as dry eye, blepharitis, and neurotrophic keratopathies represent a significant and a growing therapeutic challenge. The basis of this expanding prevalence is multifactorial and may due to issues such as an aging population, an increasing use of video display terminals, and increases in frequency of refractive surgeries. The growing incidence of diseases such as diabetes may also be a contributing factor. Current treatments for ocular surface disease include artificial tears, lubricants, tear duct plugs, steroids, antibiotics, cyclosporine, scleral lenses, and serum tears. Treatment choices depend on the type and severity of the disease, but in general positive outcomes are limited because many of these treatments do not fully address the underlying disease process or promote mechanisms that facilitate long-term wound repair. From minor corneal injuries to more severe inflammatory-mediated pathologies, clinicians need agents that promote corneal healing and reduce the inflammatory response to prevent visual disturbances and improve quality of life. A focus on treatments that reduce the inflammatory responses while accelerating corneal epithelial growth would represent a major step forward from current treatment options. Increasing evidence suggests that thymosin beta 4 (Tß4), a naturally occurring polypeptide, can elicit this spectrum of therapeutic responses: a rapid corneal reepithelialization and a reduction in corneal inflammation. This chapter serves as a review of standard therapies as well as recent advancements in the development of newer therapies that includes the use of Tß4 that is proving to be an exciting new agent for the management of ocular surface disease.

Effects of amelogenins on angiogenesis-associated processes of endothelial cells.

OBJECTIVE: To study the effects of an amelogenin mixture on integrin-dependent adhesion, DNA synthesis and apoptosis of cultured human dermal microvascular endothelial cells and angiogenesis in an organotypic assay. METHOD: Immobilised antibodies against specific integrins (alpha-1, alpha-2, alpha-3, alpha-4, alpha-5, alpha-v, ß1, ß2, ß3, ß4, ß6, alpha-vß3, alpha-vß5 and alpha-5ß1) were used to capture treated human dermal microvascular endothelial cells, which were detected colourimetrically. DNA synthesis of the cells was monitored by 5-bromo-2'- deoxyuridine incorporation and apoptosis by a TdT-mediated dUTP nick-end labelling technique. Tubule formation from aortic arches of 13-d-old chick embryos were followed over 48h. RESULTS: The amelogenin mixture increased microvessel outgrowth by 76% (p < 0.01, n=12) from the aortic explants. Also, amelogenins increased the adhesion (p < 0.01, n = 5) by multiple angiogenesis associated integrin subunits and alpha-vß3, alpha-vß5 and alpha-5ß1 heterodimers on human dermal microvascular endothelial cells at a non-mitogenic concentration (100 µg/ml). Conversely, amelogenins at 1,000 µg/ml decreased microvessel formation possibly due to attenuation of corresponding integrins despite increasing (p < 0.001, n = 8) DNA synthesis. No significant apoptosis was detected in human dermal microvascular endothelial cells cultured on Matrigel with and without amelogenins. CONCLUSION: Increased surface expression of integrins on endothelial cells may contribute to the proangiogenic property of amelogenins.

Advancing science and technology via 3D culture on basement membrane matrix.

Many cells in tissues are in contact with a highly specialized extracellular matrix, termed the basement membrane. Basement membranes have certain common components, including collagen IV, laminins, heparan sulfate proteoglycans, and growth factors which have a wide variety of biological activities. Extracts of basement membrane-rich tissue have yielded material suitable for studying cell-basement membrane interactions. Cells cultured in a 3D basement membrane matrix allow the in vitro modeling of cell behavior, including differentiation, apoptosis, steps in capillary formation, cancer growth, invasion, etc. It has also led to the development of widely used assays for invasion and angiogenesis and more recently for tumor cell dormancy. Importantly, stem cell culture in 3D basement membrane matrices has provided important advances that allow for expansion of these cells in feeder layer-free cultures and for studying their differentiation. 3D basement membrane culture has allowed the molecular dissection of pathways and genes important in differentiation, aided in the identification of progenitor cells, and led to the development of tissue constructs which may be models for regenerative medicine. This review will outline how this technology has led to important research assays and findings that have advanced our understanding of tissue development and disease and aided in the preclinical development of various therapeutics.

C16 laminin peptide increases angiotropic extravascular migration of human melanoma cells in a shell-less chick chorioallantoic membrane assay.

BACKGROUND: As distinct from intravascular dissemination, extravascular migratory metastasis (EVMM) has been described as a potential additional mechanism of melanoma spread in which tumour cells migrate along the external surfaces of vessels. Recent experimental studies strongly suggest a correlation of angiotropism of melanoma cells with EVMM. Angiotropic melanoma cells are linked to the endothelium by an amorphous matrix confirmed to contain laminin. OBJECTIVES: To investigate whether laminin plays a role in this extravascular mechanism of tumour spread. METHODS: We tested the effect of the C16 laminin peptide on melanoma spread in a shell-less chick chorioallantoic membrane model. RESULTS: After 3 days, green fluorescent protein-expressing melanoma cells were observed spreading along or in the immediate proximity of vessels. The C16 laminin peptide significantly lengthened the distance of extravascular, angiotropic migration of melanoma cells. Histopathology confirmed the angiotropism of melanoma cells without intravasation, compatible with that observed with human angiotropic melanoma. CONCLUSIONS: The results of this study suggest that the C16 laminin gamma1 chain peptide has angiotropic, extravascular migration-promoting activity on human melanoma cells, and might be a molecular target for preventing melanoma metastasis.

Association between increased CCL2 (MCP-1) expression in lesions and persistence of disease activity in giant-cell arteritis.

OBJECTIVE: Patients with giant-cell arteritis (GCA) usually respond dramatically to corticosteroid treatment. However, recurrences are frequent and corticosteroid requirements are highly variable among patients. The aim of our study was to identify genes potentially involved in disease persistence. METHODS: Gene expression was explored with cDNA arrays in temporal artery biopsies from six GCA patients with relapsing disease and six patients who easily achieved sustained remission. Differentially expressed genes of interest were subsequently analysed by quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry in temporal artery biopsies from 35 patients with biopsy-proven GCA and nine controls. RESULTS: CCL2 (MCP-1) was up-regulated in temporal artery samples from relapsing individuals. In the extended series of patients, CCL2 mRNA concentration in lesions was significantly higher than in controls (31 +/- 15.6 vs 0.44 +/- 0.10, P = 0.0001). In addition, CCL2 was more abundant in patients who experienced two or more relapses during the first year compared with those who endured sustained remission (127 +/- 82 vs 11 +/- 5.5, P = 0.0233) and correlated with the cumulated prednisolone dose (R = 0.533, P = 0.0024). CCL2 mRNA concentration correlated with IL-1beta (R = 0.45, P = 0.02), tumour necrosis factor-alpha (TNF-alpha) (R = 0.47, P = 0.013) and IL-6 (R = 0.52, P = 0.0053) mRNA. However, circulating CCL2 determined by ELISA was decreased in patients with strong systemic inflammatory response, suggesting that reduction in circulating CCL2 may reinforce the local gradient in lesions. CONCLUSION: Increased CCL2 (MCP-1) expression in lesions is associated with persistence of disease activity in GCA.

Amelogenin-mediated regulation of osteoclastogenesis, and periodontal cell proliferation and migration.

We previously reported that amelogenin isoforms M180 and leucine-rich amelogenin peptide (LRAP) are expressed in the periodontal region, and that their absence is associated with increased cementum defects in amelogenin-knockout (KO) mice. The aim of the present study was to characterize the functions of these isoforms in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament cells. The co-cultures of wild-type (WT) osteoclast progenitor and KO cementoblast/periodontal ligament cells displayed more tartrate-resistant acid phosphatase (TRAP)-positive cells than the co-cultures of WT cells. The addition of LRAP to both co-cultures significantly reduced RANKL expression and the TRAP-positive cells. Proliferation and migration rates of the KO cementoblast/periodontal ligament cells were lower than those of WT cells and increased with the addition of either LRAP or P172 (a porcine homolog of mouse M180). Thus, we demonstrate the regulation of osteoclastogenesis by LRAP, and the proliferation and migration of cementoblast/periodontal ligament cells by LRAP and P172.

Thymosin beta4 promotes angiogenesis, wound healing, and hair follicle development.

New blood vessel formation is important in many physiological process, including development, wound repair, and tumor growth. In aged animals, angiogenesis is reduced resulting in poor wound healing. We have identified a novel small molecule, thymosin beta(4), that promotes angiogenesis and wound repair in both normal and aged rodents. It also promotes hair growth in normal and aged rodents. It acts by increasing angiogenesis and cell migration and is currently in clinical trials for wound repair.

NOVH increases MMP3 expression and cell migration in glioblastoma cells via a PDGFR-alpha-dependent mechanism.

Nephroblastoma overexpressed gene (NOV) is highly expressed in the nervous system. We investigated its biological activity by expressing the human NOV gene (NOVH) in a human glioblastoma cell line that is negative for NOVH and by analyzing four clones with different levels of NOVH expression. There was no difference in cell proliferation between the NOVH-expressing cell lines, but there was increased cell adhesion and migration that correlated with increasing NOVH expression. Gene expression profiling was used to investigate the mechanisms by which NOVH expression regulated cell activity. We identified two induced genes in NOVH-expressing cells that are involved in cell migration: matrix metalloprotease (MMP)3 and platelet-derived growth factor receptor (PDGFR)-alpha. Our studies show that PDGFR-alpha induced MMP3 gene expression and increased cell proliferation and cell migration upon stimulation by platelet-derived growth factor (PDGF)-AA. We also show that the induction of MMP3 in cells expressing NOVH is potentiated by either cell density, serum, or PDGF-BB. Thus, expression of NOVH in glioblastoma cells triggers a cascade of gene expression resulting in increased cell adhesion and migration.

Biological response of chondrocytes to hydrogels.

Primary bovine chondrocytes were encapsulated in alginate and alginate combined with cartilage matrix extract, Cartrigel, for the purpose of cartilage tissue engineering. The cell constructs were incubated in vitro and gene expression of cartilage-specific extracellular matrix molecules was quantitated and localized with in situ hybridization with a decrease in expression observed in the alginate-Cartrigel constructs. Further understanding of cell response to scaffolds will allow rational design and development of hydrogels for cartilage tissue engineering.

Laminin gamma 1 chain peptide, C-16 (KAFDITYVRLKF), promotes migration, MMP-9 secretion, and pulmonary metastasis of B16-F10 mouse melanoma cells.

Laminin-1, a heterotrimer of alpha 1, beta 1, and gamma 1 chains specific to basement membrane, promotes cell adhesion and migration, proteinase secretion and metastases of tumour cells. Several active sites on the alpha 1 chain have been found to promote B16-F10 melanoma lung colonisation and here we have determined whether additional tumour promoting sites exist on the beta 1 and gamma 1 chains. Recently, we have identified novel cell adhesive peptides derived from laminin beta 1 and gamma 1 chains by systematic screening of synthetic peptides. Nine beta 1 peptides and seven gamma 1 peptides active for cell adhesion were tested for their effects on experimental pulmonary metastases of B16-F10 mouse melanoma cells in vivo. The most active adhesive peptide derived from the gamma 1 chain globular domain, C-16 (KAFDITYVRLKF), significantly enhanced pulmonary metastases of B16-F10 cells, whereas no other peptides showed enhancement. C-16 also stimulated migration of B16-F10 cells in the Boyden chamber assay in vitro. Furthermore, C-16 significantly induced the production of MMP-9 from B16-F10 cells. These results suggest that this specific laminin gamma 1 chain peptide has a metastasis-promoting activity and might be a new molecular target of anti-cancer treatment.

Identification of homologous biologically active sites on the N-terminal domain of laminin alpha chains.

Laminin, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha, beta, and gamma chains. To date, five different alpha chains have been identified. N-terminal domain VI in the alpha1 chain has various biological activities. Here we screened biologically active sequences on domain VI of the laminin alpha2, alpha3, and alpha5 chains using a large number of overlapping peptides. HT-1080 human fibrosarcoma cell attachment to the peptides was evaluated using peptide-coated plastic plates and peptide-conjugated Sepharose beads. We identified four cell adhesive sequences from laminin alpha2 chain domain VI, two sequences from the alpha3 chain, and two sequences from the laminin alpha5 chain. Sequences homologous to A13 (RQVFQVAYIIIKA, alpha1 chain 121-133) on all the alpha chains (FQIAYVIVKA, alpha2 chain 130-139; GQLFHVAYILIKF, alpha3 chain 96-108; FHVAYVLIKA, alpha5 chain 74-83) showed strong cell attachment activity. A5-16 (LENGEIVVSLVNGR, alpha5 chain 147-160) showed the strongest cell attachment activity in the plate assay, and the homologous peptide in the alpha3 chain promoted similar strong cell attachment activity. A5-16 and its homologous peptide in the alpha2 chain promoted moderate cell attachment, while the homologous peptide to A5-16 in the alpha1 chain did not show activity. A2-7 (SPSIKNGVEYHYV, alpha2 chain 108-120) showed cell attachment activity only in the plate assay, but homologous sequences in the alpha1, alpha3, and alpha5 chains did not promote activity. A2-7 promoted endothelial cell sprouting from aortic rings in vitro and melanoma colonization to murine lungs in vivo. However, none of the homologous peptides of A2-7 promoted experimental pulmonary metastasis by B16-BL6 melanoma cells. These results indicate that there are chain-specific active sites in domain VI of the laminin alpha chains, some of which contain conserved activities.

Evaluation of the chemopreventive potential of retinoids using a novel in vitro human prostate carcinogenesis model.

The prevalence of prostatic intraepithelial neoplasia (PIN) and latent prostatic carcinoma, representing multiple steps in carcinogenesis and progression to invasive carcinoma, makes them relevant targets for prevention. A unique family of human prostate epithelial cell lines, which mimic steps in prostate carcinogenesis and progression, were used to evaluate the chemopreventive potential of all-trans-retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4-HPR). The effects of RA and 4-HPR on anchorage-dependent growth of an immortalized, non-tumorigenic cell line RWPE-1 and two tumorigenic cell lines, WPE1-NB14 and WPE1-NB11, derived from RWPE-1 by exposure to N-methyl-N-nitrosourea (MNU), were examined. Both tumorigenic cell lines grow more rapidly than the parent RWPE-1 cell line in monolayer culture. Further, while RWPE-1 cells do not form colonies in agar, both tumorigenic cell lines do, with a colony forming efficiency (CFE) of 1.85 and 2.04% for WPE1-NB14 and WPE1-NB11 cells, respectively. Both RA and 4-HPR inhibited anchorage-dependent growth of all cell lines and anchorage-independent growth of WPE1-NB14 and WPE1-NB11 cells, in a dose-dependent manner, however, 10 times more RA than 4-HPR was required to produce the same effect. RWPE-1 cells are not invasive but WPE1-NB11 cells are significantly more invasive than WPE1-NB14 cells. Both RA and 4-HPR inhibited invasion in vitro by WPE1-NB11 and WPE1-NB14 cells where the more malignant WPE1-NB11 cells showed greater inhibition of invasion by 4-HPR than by RA. Overall, 4-HPR was more effective than RA in inhibiting growth and invasion but the response varied amongst the cell lines. These three cell lines mimic progressive steps in carcinogenesis and progression, from immortalized, non-tumorigenic RWPE-1 cells, to the less malignant WPE1-NB14 to the more malignant WPE1-NB11 cells, and provide powerful models for studies on secondary and tertiary prevention, i.e. promotion and progression stages, respectively, of prostate cancer.

The role of alpha 6 beta 1 integrin and EGF in normal and malignant acinar morphogenesis of human prostatic epithelial cells.

Complex multiple interactions between cells and extracellular matrix occur during acinar morphogenesis involving integrin receptors and growth factors. Changes in these interactions occur during carcinogenesis as cells progress from a normal to a malignant, invasive phenotype. We have developed human prostatic epithelial cell lines of the same lineage, which represent multiple steps in carcinogenesis, similar to prostatic intraepithelial neoplasia and subsequent tumor progression. The non-tumorigenic, RWPE-1 and the tumorigenic WPE1-NB27 and WPE1-NB26 cell lines were used to examine their ability to undergo acinar morphogenesis in a 3-D cell culture model and its relationship to invasion, integrin expression and EGF presence. An inverse relationship between the degree of acinar formation and invasive ability was observed. The non-tumorigenic, non-invasive RWPE-1 and the low tumorigenic, low invasive, WPE1-NB27 cells show high and decreased acinar forming ability, respectively, while the more invasive WPE1-NB26 cells show a loss of acinar formation. While RWPE-1 acini show basal expression of alpha 6 beta 1 integrin, which correlates with their ability to polarize and form acini, WPE1-NB27 cells lack alpha 6 but show basal, but weaker expression of beta 1 integrin. WPE1-NB26 cells show loss alpha 6 and abnormal, diffused beta 1 integrin expression. A dose-dependent decrease in acinar formation was observed in RWPE-1 cells when cell proliferation was induced by EGF. Anti-functional antibody to EGF caused an increase in acinar formation in RWPE-1 cells. These results suggest that malignant cells lose the ability to undergo acinar morphogenesis and that the degree of this loss appears to be related to invasive ability, EGF levels and alterations in laminin-specific integrin expression. This model system mimics different steps in prostate carcinogenesis and has applications in the secondary and tertiary prevention of prostate cancer.

An angiogenic laminin site and its antagonist bind through the alpha(v)beta3 and alpha5beta1 integrins.

Angiogenesis is important for wound healing, tumor growth, and metastasis. Endothelial cells differentiate into capillary-like structures on a laminin-1-rich matrix (Matrigel). We previously identified 20 angiogenic sites on laminin-1 (alpha1beta1gamma1) by screening 559 overlapping synthetic peptides. C16, the most potent gamma1 chain peptide, blocked laminin-1-mediated adhesion and was the only gamma1 chain peptide to block attachment to both collagen I and fibronectin. This suggested that C16 was acting via a receptor common to these substrates. We demonstrated that C16 is angiogenic in vivo. Affinity chromatography identified the integrins alpha5beta1 and alpha(v)beta3 as surface receptors. Blocking antibodies confirmed the role of these receptors in C16 adhesion. C16 does not contain an RGD sequence and, as expected, an RGD-containing peptide did not block C16 adhesion nor did C16 act via MAP kinase phosphorylation. Furthermore, we identified a C16 scrambled sequence, C16S, which antagonizes the angiogenic activity of bFGF and of C16 by binding to the same receptors. Because the laminin gamma1 chain is ubiquitous in most tissues, C16 is likely an important functional site. Since the biological activity of C16 is blocked by a scrambled peptide, C16S may serve as an anti-angiogenic therapeutic agent.

Eotaxin (CCL11) induces in vivo angiogenic responses by human CCR3+ endothelial cells.

Chemokines are attractants and regulators of cell activation. Several CXC family chemokine members induce angiogenesis and promote tumor growth. In contrast, the only CC chemokine, reported to play a direct role in angiogenesis is monocyte-chemotactic protein-1. Here we report that another CC chemokine, eotaxin (also known as CCL11), also induced chemotaxis of human microvascular endothelial cells. CCL11-induced chemotactic responses were comparable with those induced by monocyte-chemotactic protein-1 (CCL2), but lower than those induced by stroma-derived factor-1alpha (CXCL12) and IL-8 (CXCL8). The chemotactic activity was consistent with the expression of CCR3, the receptor for CCL11, on human microvascular endothelial cells and was inhibited by mAbs to either human CCL11 or human CCR3. CCL11 also induced the formation of blood vessels in vivo as assessed by the chick chorioallantoic membrane and Matrigel plug assays. The angiogenic response induced by CCL11 was about one-half of that induced by basic fibroblast factor, and it was accompanied by an inflammatory infiltrate, which consisted predominantly of eosinophils. Because the rat aortic sprouting assay, which is not infiltrated by eosinophils, yielded a positive response to CCL11, this angiogenic response appears to be direct and is not mediated by eosinophil products. This suggests that CCL11 may contribute to angiogenesis in conditions characterized by increased CCL11 production and eosinophil infiltration such as Hodgkin's lymphoma, nasal polyposis, endometriosis, and allergic diathesis.

Stimulation of tumor growth by human soluble intercellular adhesion molecule-1.

Because serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) are elevated in cancer and sICAM-1 is angiogenic, we tested the ability of sICAM-1 to promote tumor growth. Our preliminary experiments showed that exogenous sICAM-1 significantly stimulated the growth of human tumors in vivo. Human fibrosarcoma transfectants, which express ICAM-1, produce ICAM-1 on the cell surface and release sICAM-1 into the medium without any apparent effect on cell growth in vitro. We found that conditioned medium from sense ICAM-1 transfectants compared with mock or antisense ICAM-1 transfectants stimulates endothelial cell migration in vitro and neovascularization in the chick chorioallantoic membrane assay. Tumor cells transfected with sense constructs form faster growing tumors than mock- and antisense-transfected cells in both chick embryos and nude mice models. Serum levels of human sICAM-1 from nude mice bearing sense ICAM-1 transfectants correlate positively with tumor weight. Sense ICAM-1 transfectants are more proliferative and induce more blood vessel formation than mock and antisense transfectants in nude mice. Because expression of ICAM-1 does not affect tumor cell growth in vitro, the angiogenic activity of sICAM-1 produced by sense ICAM-1 transfectants may be involved in the stimulation of tumor growth. Therefore, sICAM-1 may perform dual functions that are essential for tumor growth: angiogenesis and escape from immune surveillance.

Role of basement membrane in tumor growth and metastasis.

The basement membrane is a thin extracellular matrix produced by epithelial and endothelial cells. It is biologically active for normal epithelial cell differentiation. Basement membrane promotes the growth of tumor cells in vitro and in vivo when coinjected. Laminin, the major biologically active component, also increases tumor growth and the malignant phenotype by promoting increased cell growth and protease activity. Using systematic peptide screening with synthetic peptides covering the entire laminin molecule, several active sites in laminin have been identified that regulate tumor growth and metastasis.