Interference in melanoma CD248 function reduces vascular mimicry and metastasis.

BACKGROUND: Tumor vascular mimicry is an emerging issue that affects patient survival while having no treatment at the current moment. Despite several factors implicated in vascular mimicry, little is known about stromal factors that modulate tumor microenvironment and shape malignant transformation. CD248, a type-I transmembrane protein dominantly expressed in stromal cells, mediates the interaction between cells and extracellular matrix proteins. CD248 protein expression is associated with the metastatic melanoma phenotype and promotes tumor progression in the stromal cells. This study aimed to explore the cell-autonomous effects of CD248 in melanoma vascular mimicry to aid cancer therapy development. METHODS: Loss-of-function approaches in B16F10 melanoma cells were used to study the cell-autonomous effects of CD248 on cell adhesion, migration, proliferation, and vascular mimicry. A solid-phase binding assay was performed to identify the interaction between CD248 and fibronectin. Horizontal and vertical cell migration assays were performed to analyze cell migration activity, and cell-patterned network formation on Matrigel was used to evaluate vascular mimicry activity. Recombinant CD248 (rCD248) proteins were generated, and whether rCD248 interfered with melanoma CD248 functions was evaluated in vitro. An experimental lung metastasis mouse model was used to investigate the effect of rCD248 treatment in vivo. RESULTS: CD248 protein expression in melanoma cells was increased by a fibroblast-conditioned medium. Knockdown of CD248 expression significantly decreased cell adhesion to fibronectin, cell migration, and vascular mimicry in melanoma cells. The lectin domain of CD248 was directly involved in the interaction between CD248 and fibronectin. Furthermore, rCD248 proteins containing its lectin domain inhibited cell adhesion to fibronectin and slowed down cell migration and vascular mimicry. Treatment with rCD248 protein could reduce pulmonary tumor burden, accompanied by a reduction in vascular mimicry in mice with melanoma lung metastasis. CONCLUSION: CD248 expression in melanoma cells promotes malignant transformation by increasing the activity of cell adhesion, migration, and vascular mimicry, whereas rCD248 protein functions as a molecular decoy interfering with tumor-promoting effects of CD248 in melanoma cells.

Recombinant thrombomodulin lectin-like domain attenuates Porphyromonas gingivalis lipopolysaccharide-induced osteoclastogenesis and periodontal bone resorption.

BACKGROUND: Evidence demonstrates that the thrombomodulin (TM) lectin domain (TMD1) exerts anti-inflammatory functions. Lipopolysaccharides derived from Porphyromonas gingivalis (Pg-LPS) are considered a major pathogenic factor for chronic periodontitis, promoting inflammation, osteoclastogenesis and alveolar bone resorption. Herein, we aimed to evaluate the potential therapeutic effect of recombinant TMD1 (rTMD1) in suppression of Pg-LPS-induced osteoclastogenesis and periodontal bone loss. METHODS: In vitro, the effects of Pg-LPS, tumor necrosis factor (TNF)-α and rTMD1 on osteoclast differentiation were investigated using receptor activator of nuclear factor-κB ligand (RANKL)-stimulated RAW 264.7 macrophages. In vivo, the effects of rTMD1 treatment were evaluated in a model of experimental periodontitis induced by direct injection of Pg-LPS into the vestibular gingiva. RESULTS: Administration of Pg-LPS to RANKL-stimulated RAW 264.7 macrophages resulted in upregulation of CD86 and osteoclast marker (eg, Dc-stamp and Trap) gene expression and increase of pro-inflammatory cytokine production (e.g., TNF-α) during osteoclast differentiation, and rTMD1 can attenuate these effects. Also, rTMD1 inhibited Pg-LPS-enhanced in vitro bone resorption in a dose-dependent manner. Moreover, TNF-α promoted phosphorylation of p38 and ERK during osteoclast differentiation, and the signal activation can be inhibited by rTMD1. Finally, treatment with rTMD1 hindered Pg-LPS-induced alveolar bone loss in experimental periodontitis in mice. CONCLUSION: Our study demonstrated that rTMD1 attenuates Pg-LPS-enhanced M1 macrophage polarization, osteoclastogenesis and periodontal bone resorption and thus holds therapeutic promise for periodontitis.

Tumor Endothelial Marker 1 (TEM1/Endosialin/CD248) Enhances Wound Healing by Interacting with Platelet-Derived Growth Factor Receptors.

Tumor endothelial marker 1 (TEM1), also known as endosialin or CD248, is a type I transmembrane glycoprotein containing a C-type lectin-like domain. It is highly expressed in pericytes and fibroblasts. Dermal fibroblasts play a pivotal role during cutaneous wound healing, especially in the proliferative phase. However, the physiological function of TEM1 in wound healing is still undetermined. During the process of wound healing, the expression of both TEM1 and platelet-derived growth factor (PDGF) receptor α was highly upregulated in myofibroblasts. In vivo, fibroblast activation and collagen deposition in granulation tissues were attenuated, and wound healing was retarded in TEM1-deleted mice. In vitro, the migration, adhesion, and proliferation of NIH3T3 cells were suppressed following TEM1 knockdown by short hairpin RNA. In PDGF-BB-treated NIH3T3 cells, the downstream signal and mitogenic, and chemoattractive effects were inhibited by TEM1 knockdown. In addition, TEM1 and PDGF receptor α were colocalized in subcellular organelles in fibroblasts, and the association of TEM1 and PDGF receptor α was demonstrated by coimmunoprecipitation. In summary, these findings suggested that TEM1, in combination with PDGF receptor α, plays a critical role in wound healing by enhancing the mitogenic and chemoattractive effects of PDGF-BB and collagen deposition in myofibroblasts.

Recombinant adeno-associated virus vector carrying the thrombomodulin lectin-like domain for the treatment of abdominal aortic aneurysm.

BACKGROUND AND AIMS: Thrombomodulin (TM), through its lectin-like domain (TMD1), sequesters proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that sustains inflammation and tissue damage. Our previous study demonstrated that short-term treatment with recombinant TM containing all the extracellular domains (i.e., rTMD123) inhibits HMGB1-RAGE signaling and confers protection against CaCl2-induced AAA formation. In this study, we attempted to further optimize TM domains, as a potential therapeutic agent for AAA, using the recombinant adeno-associated virus (AAV) vector. METHODS: The therapeutic effects of recombinant TMD1 (rTMD1) and recombinant AAV vectors carrying the lectin-like domain of TM (rAAV-TMD1) were evaluated in the CaCl2-induced AAA model and angiotensin II-infused AAA model, respectively. RESULTS: In the CaCl2-induced model, treatment with rTMD1 suppressed the tissue levels of HMGB1 and RAGE, macrophage accumulation, elastin destruction and AAA formation, and the effects were comparable to a mole-equivalent dosage of rTMD123. In the angiotensin II-infused model, a single intravenous injection of rAAV-TMD1 (1011 genome copies), which resulted in a persistently high serum level of TMD1 for at least 12 weeks, effectively attenuated AAA formation with suppression of HMGB1 and RAGE levels and inhibition of proinflammatory cytokine production, macrophage accumulation, matrix metalloproteinase activities and oxidative stress in the aortic wall. CONCLUSIONS: These findings corroborate the therapeutic potential of the TM lectin-like domain in AAA. The attenuation of angiotensin II-infused AAA by one-time delivery of rAAV-TMD1 provides a proof-of-concept validation of its application as potential gene therapy for aneurysm development.

Development of Recombinant Adeno-Associated Virus Serotype 2/8 Carrying Kringle Domains of Human Plasminogen for Sustained Expression and Cancer Therapy.

Angiostatin and other plasminogen derivatives exhibit antitumor activities directly or indirectly, have demonstrated promising anticancer effects in preclinical studies, but have mostly failed in clinical trials partly due to their short serum half-lives. Our previous studies demonstrated that recombinant human plasminogen kringle 1-5 (K1-5) has superior antitumor activity compared with angiostatin. In addition, optimization of recombinant K1-5 with three amino acid substitutions enhances its antitumor effect. The current study was thus undertaken to evaluate prolonged expression of optimized K1-5 as cancer gene therapy. The recombinant adeno-associated virus (AAV) vector was used to express a secreted form of the optimized K1-5 (AAV-sK15tm) to improve its pharmacokinetic profile, which was considered to be the hurdle in angiostatin treatment of cancer. We successfully generated high-titer recombinant AAV vectors and observed sustained transgene expression for 567 days after a single injection of virus. The treated animals did not display any visible signs of abnormalities and showed normal serum biochemistry. The therapeutic potential of this treatment modality was demonstrated by both a strong inhibition of lung metastasis in the mouse B16F10 melanoma model and significant growth retardation of Lewis lung carcinoma xenografts in C57BL/6N mice as well as human A2058 melanoma xenografts in NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Taken together, our results suggested that AAV-sK15tm produced long-term suppressive effects on cancer growth in vivo and should warrant serious consideration for clinical development.

Thrombomodulin promotes diabetic wound healing by regulating toll-like receptor 4 expression.

Keratinocyte-expressed thrombomodulin (TM) and the released soluble TM (sTM) have been demonstrated to promote wound healing. However, the effects of high glucose on TM expression in keratinocytes and the role of TM in diabetic ulcer remain unclear. In this study, we demonstrated that expressions of TM and Toll-like receptor 4 (TLR4) were both downregulated in high-glucose cultured human keratinocytes and in skin keratinocytes of diabetic patients. In addition, the wound-triggered upregulation of TM and sTM production was abolished in both high-glucose cultured human keratinocytes and streptozotocin-induced diabetic mouse skin. Furthermore, supplementation of recombinant sTM could increase TLR4 expression and promote cutaneous wound healing in both high-glucose cultured human keratinocytes and diabetic mice. However, in Tlr4-deleted mice, which exhibited delayed wound healing, the therapeutic benefit of recombinant sTM was abrogated. Moreover, our results showed that tumor necrosis factor-α (TNF-α) expression in keratinocytes was dose-dependently upregulated by glucose, and TNF-α treatment downregulated the expression of TM and TLR4. Taken together, high-glucose environment reduces the expression of TM and TLR4 in keratinocytes possibly through the action of TNF-α, and recombinant sTM can increase the TLR4 expression and promote wound healing under diabetic condition.

FGFR1 mediates recombinant thrombomodulin domain-induced angiogenesis.

AIMS: The recombinant epidermal growth factor-like domain plus the serine/threonine-rich domain of thrombomodulin (rTMD23) promotes angiogenesis and accelerates the generation of activated protein C (APC), which facilitates angiogenesis. The aim of this study was to elucidate the molecular mechanisms underlying the angiogenic activity of rTMD23. METHODS AND RESULTS: We prepared rTMD23 and its mutants that did not possess the ability to promote APC generation and investigated their angiogenic activities in vitro and in vivo. rTMD23 mutants promoted proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro and induced neovascularization in vivo; these effects were similar to those exerted by wild-type rTMD23. To investigate its interaction with rTMD23, Type I fibroblast growth factor receptor (FGFR1) was precipitated along with syndecan-4 by rTMD23-conjugated Sepharose in human umbilical vein endothelial cells and FGFR1-expressing human embryonic kidney 293 cells. Additionally, the kinetics of the interaction between rTMD23 and FGFR1 were analysed using surface plasmon resonance. rTMD23-induced FGFR1 activation and tube formation were inhibited by an FGFR1-specific tyrosine kinase inhibitor, PD173074, or by knockdown of FGFR1 using siRNA technology. We observed an improvement in rat hindlimb recovery in an ischaemic model following rTMD23 treatment, and this was associated with increased neovascularization and FGFR1 phosphorylation. CONCLUSION: rTMD23 induced angiogenesis via FGFR1, a process that is independent of the APC pathway.

RHBDL2 is a critical membrane protease for anoikis resistance in human malignant epithelial cells.

Anoikis resistance allows metastatic tumor cells to survive in a homeless environment. Activation of epithelial growth factor receptor (EGFR) signaling is one of the key mechanisms for metastatic tumor cells to resist anoikis, yet the regulation mechanisms of homeless-triggered EGFR activation in metastatic tumor cells remain unclear. Rhomboid-like-2 (RHBDL2), an evolutionally conserved intramembrane serine protease, can cleave the EGF ligand and thus trigger EGFR activation. Herein, we demonstrated that RHBDL2 overexpression in human epithelial cells resulted in promotion of cell proliferation, reduction of cell adhesion, and suppression of anoikis. During long-term suspension cultures, increased RHBDL2 was only detected in aggressive tumor cell lines. Treatment with the rhomboid protease inhibitor or RHBDL2 shRNA increased cleaved caspase 3, a marker of apoptosis. Finally, inhibition of EGFR activation increased the cleaved caspase 3 and attenuated the detachment-induced focal adhesion kinase phosphorylation. Taken together, these findings provide evidence for the first time that RHBDL2 is a critical molecule in anoikis resistance of malignant epithelial cells, possibly through the EGFR-mediated signaling. Our study demonstrates RHBDL2 as a new therapeutic target for cancer metastasis.

Thrombomodulin functions as a plasminogen receptor to modulate angiogenesis.

Urokinase-type plasminogen activator (uPA) activates plasminogen (Plg) through a major pericellular proteolytic system involved in cell migration and angiogenesis; however, the Plg receptor that participates in uPA-mediated Plg activation has not yet been identified. In this study, we demonstrated that thrombomodulin (TM), a type I transmembrane glycoprotein, is a novel Plg receptor that plays a role in pericellular proteolysis and cell migration. Plg activation at the cell surface and the extent of its cell migration- and invasion-promoting effect are cellular TM expression dependent. Direct binding of Plg and the recombinant TM extracellular domain, with a KD of 0.1-0.3 µM, was determined through surface plasmon resonance analysis. Colocalization of TM, Plg, and the uPA receptor within plasma membrane lipid rafts, at the leading edge of migrating endothelial cells, was demonstrated and was also shown to overlap with areas of major pericellular proteolysis. Moreover, the roles of TM and Plg in neoangiogenesis were demonstrated in vivo through the skin wound-healing model. In conclusion, we propose that TM is a novel Plg receptor that regulates uPA/uPA receptor-mediated Plg activation and pericellular proteolysis within lipid rafts at the leading edge of migrating cells during angiogenesis.

Recombinant human thrombomodulin suppresses experimental abdominal aortic aneurysms induced by calcium chloride in mice.

OBJECTIVE: To investigate whether recombinant thrombomodulin containing all the extracellular domains (rTMD123) has therapeutic potential against aneurysm development. SUMMARY BACKGROUND DATA: The pathogenesis of abdominal aortic aneurysm (AAA) is characterized by chronic inflammation and proteolytic degradation of extracellular matrix. Thrombomodulin, a transmembrane glycoprotein, exerts anti-inflammatory activities such as inhibition of cytokine production and sequestration of proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that may sustain inflammation and tissue damage. METHODS: The in vivo effects of treatment and posttreatment with rTMD123 on aortic dilatation were measured using the CaCl2-induced AAA model in mice. RESULTS: Characterization of the CaCl2-induced model revealed that HMGB1 and RAGE, both localized mainly to macrophages, were persistently upregulated during a 28-day period of AAA development. In vitro, rTMD123-HMGB1 interaction prevented HMGB1 binding to macrophages, thereby prohibiting activation of HMGB1-RAGE signaling in macrophages. In vivo, short-term treatment with rTMD123 upon AAA induction suppressed the levels of proinflammatory cytokines, HMGB1, and RAGE in the aortic tissue; reduced the infiltrating macrophage number; and finally attenuated matrix metalloproteinase production, extracellular matrix destruction, and AAA formation without disturbing vascular calcification. Consistently, posttreatment with rTMD123 seven days after AAA induction alleviated vascular inflammation and retarded AAA progression. CONCLUSIONS: These data suggest that rTMD123 confers protection against AAA development. The mechanism of action may be associated with reduction of proinflammatory mediators, blockade of macrophage recruitment, and suppression of HMGB1-RAGE signaling involved in aneurysm formation and downstream macrophage activation.

Thrombomodulin regulates keratinocyte differentiation and promotes wound healing.

The membrane glycoprotein thrombomodulin (TM) has been implicated in keratinocyte differentiation and wound healing, but its specific function remains undetermined. The epidermis-specific TM knockout mice were generated to investigate the function of TM in these biological processes. Primary cultured keratinocytes obtained from TM(lox/lox); K5-Cre mice, in which TM expression was abrogated, underwent abnormal differentiation in response to calcium induction. Poor epidermal differentiation, as evidenced by downregulation of the terminal differentiation markers loricrin and filaggrin, was observed in TM(lox/lox); K5-Cre mice. Silencing TM expression in human epithelial cells impaired calcium-induced extracellular signal-regulated kinase pathway activation and subsequent keratinocyte differentiation. Compared with wild-type mice, the cell spreading area and wound closure rate were lower in keratinocytes from TM(lox/lox); K5-Cre mice. In addition, the lower density of neovascularization and smaller area of hyperproliferative epithelium contributed to slower wound healing in TM(lox/lox); K5-Cre mice than in wild-type mice. Local administration of recombinant TM (rTM) accelerated healing rates in the TM-null skin. These data suggest that TM has a critical role in skin differentiation and wound healing. Furthermore, rTM may hold therapeutic potential for the treatment of nonhealing chronic wounds.

The epidermal growth factor-like domain of CD93 is a potent angiogenic factor.

Human CD93, an epidermal growth factor (EGF)-like domain containing transmembrane protein, is predominantly expressed in the vascular endothelium. Studies have shown that AA4, the homolog of CD93 in mice, may mediate cell migration and angiogenesis in endothelial cells. Soluble CD93 has been detected in the plasma of healthy individuals. However, the role of soluble CD93 in the endothelium remains unclear. Recombinant soluble CD93 proteins with EGF-like domains (rCD93D123, with domains 1, 2, and 3; and rCD93D23, with domains 2 and 3) were generated to determine their functions in angiogenesis. We found that rCD93D23 was more potent than rCD93D123 in stimulating the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Production of matrix-metalloproteinase 2 increased after the HUVECs were treated with rCD93D23. Further, in a tube formation assay, rCD93D23 induced cell differentiation of HUVECs through phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase and extracellular signal-regulated kinases-1/2 signaling. Moreover, rCD93D23 promoted blood vessel formation in a Matrigel-plug assay and an oxygen-induced retinopathy model in vivo. Our findings suggest that the soluble EGF-like domain containing CD93 protein is a novel angiogenic factor acting on the endothelium.

The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen.

Lewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM-TM domain 1 (rTMD1)-in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.

Lectin-like domain of thrombomodulin binds to its specific ligand Lewis Y antigen and neutralizes lipopolysaccharide-induced inflammatory response.

Thrombomodulin (TM), a widely expressing glycoprotein originally identified in vascular endothelium, is an important cofactor in the protein C anticoagulant system. TM appears to exhibit anti-inflammatory ability through both protein C-dependent and -independent pathways. We presently have demonstrated that recombinant N-terminal lectinlike domain of TM (rTMD1) functions as a protective agent against sepsis caused by Gram-negative bacterial infections. rTMD1 caused agglutination of Escherichia coli and Klebsiella pneumoniae and enhanced the macrophage phagocytosis of these Gram-negative bacteria. Moreover, rTMD1 bound to the Klebsiella pneumoniae and lipopolysaccharide (LPS) by specifically interacting with Lewis Y antigen. rTMD1 inhibited LPS-induced inflammatory mediator production via interference with CD14 and LPS binding. Furthermore, rTMD1 modulated LPS-induced mitogen-activated protein kinase and nuclear factor-kappaB signaling pathway activations and inducible nitric oxide synthase expression in macrophages. Administration of rTMD1 protected the host by suppressing inflammatory responses induced by LPS and Gram-negative bacteria, and enhanced LPS and bacterial clearance in sepsis. Thus, rTMD1 can be used to defend against bacterial infection and inhibit LPS-induced inflammatory responses, suggesting that rTMD1 may be valuable in the treatment of severe inflammation in sepsis, especially in Gram-negative bacterial infections.