Malawer type I/V proximal humerus reconstruction after tumor resection: a systematic review.

BACKGROUND: Several reconstruction methods exist for Malawer type I/V proximal humerus reconstruction after bone tumor resection; however, no consensus has been reached regarding the preferred methods. METHODS: We conducted a literature search on various types of proximal humerus oncologic reconstruction methods. We collected data on postoperative functional outcomes assessed based on Musculoskeletal Tumor Society (MSTS) scores, 5-year reconstruction survival rates, and complications. We calculated each reconstruction's weighted mean based on the sample size and standard errors. Complications were categorized based on the Henderson classification. Based on these integrated data, our primary objective is to propose an optimal strategy for proximal humerus reconstruction after bone tumor resection. RESULTS: We examined various reconstruction techniques, including modular prosthesis (752 patients in 21 articles), osteoarticular allograft (142 patients in six articles), allograft prosthesis composites (236 patients in 12 articles), reverse shoulder total arthroplasty (141 patients in 10 articles), composite reverse shoulder total arthroplasty (33 patients in four articles), claviculo-pro-humero technique (51 patients in six articles), and cement spacer (207 patients in four articles). Weighted mean MSTS scores were: modular prosthesis (73.8%), osteoarticular allograft (74.4%), allograft prosthesis composites (79.2%), reverse shoulder total arthroplasty (77.0%), composite reverse shoulder total arthroplasty (76.1%), claviculo-pro-humero technique (75.1%), and cement spacer (69.1%). Weighted 5-year reconstruction survival rates were modular prosthesis (85.4%), osteoarticular allograft (67.6%), allograft prosthesis composites (85.2%), reverse shoulder total arthroplasty (84.1%), and cement spacer (88.0%). Reconstruction survival data was unavailable for composite reverse shoulder total arthroplasty and claviculo-pro-humero technique. Major complications included shoulder joint instability: modular prosthesis (26.2%), osteoarticular allograft (41.5%), allograft prosthesis composites (33.9%), reverse shoulder total arthroplasty (17%), composite reverse shoulder total arthroplasty (6.1%), claviculo-pro-humero technique (2.0%), and cement spacer (8.7%). Aseptic loosening of the prosthesis occurred: modular prosthesis (3.9%) and reverse shoulder total arthroplasty (5.7%). Allograft fracture was observed in 54.9% of patients with osteoarticular allograft. CONCLUSION: The complication profiles differed among reconstruction methods. Weighted mean MSTS scores exceeded 70% in all methods except cement spacer, and the 5-year reconstruction survival rate surpassed 80% for all methods except osteoarticular allograft. Proximal humerus reconstruction after bone tumor resection should consider potential complications and patients' individual factors.

Outcomes of Window Therapy with Carboplatin and Ifosfamide for Pediatric Osteosarcoma: A Case Series.

For the treatment of osteosarcoma, cisplatin (CDDP) can be substituted by carboplatin (CBDCA) to reduce toxicity. We report a single institution experience of CBDCA-based regimen. Two to three cycles of CBDCA + ifosfamide (IFO) therapy (window therapy) were administered as neoadjuvant therapy for osteosarcoma. Depending on the response of window therapy, the subsequent protocols were determined; for good responders, surgery is performed, and postoperative therapies with CBDCA + IFO, adriamycin (ADM) and high-dose methotrexate (MTX) were administered; for stable disease, the postoperative regimens were advanced before surgery, and the remaining amount of postoperative chemotherapy is deduced; for progressive disease, CBDCA-based regimen is changed to CDDP-based regimen. From 2009 to 2019, seven patients were treated with this protocol. During the window therapy, two patients (28.6%) were assessed as good responders and completed the regimen as planned. Four patients (57.1%) had stable disease, and the chemotherapy schedules were modified. One patient (14.2%) with progressive disease was shifted to the CDDP-based regimen. At final follow-up, four patients showed no evidence of disease and three patients died of the disease. Since the efficacy during window therapy was limited, a CBDCA-based regimen in the neoadjuvant setting was considered insufficient for performing adequate surgery.

Bone Metastases from Gastric Cancer Resembling Paget's Disease: A Case Report.

Systemic osteosclerotic lesions are frequently caused by multiple bone metastases or systemic metabolic disorders. However, bone metastasis from gastric cancer is rare. Herein, we describe such a case, with radiographic and clinical findings resembling Paget's disease. The patient was an 80-year-old Japanese woman with a history of early gastric cancer, treated by partial gastrectomy 2 years prior. The patient sought medical care for chronic low back pain. On imaging, systemic sclerotic lesions were observed throughout the spine and pelvis, with an increase in bone mineral density from 0.86 g/cm3 (2 years prior) to 1.38g/cm3 (current visit) in the lumbar spine. Elevated serum levels of osteoblastic and osteolytic markers were identified. A bone biopsy was used to confirm the diagnosis of metastatic gastric cancer. The patient was treated with TS-1 and denosumab, with normalization of abnormal metabolic markers and alleviation of the back pain. Bone metastasis is reported in only 10% of cases of gastric cancer and, thus, is relatively rare. Therefore, our case of gastric cancer recurrence presenting with mixed osteoblastic and osteolytic bone lesions similar to Paget's disease is relevant to the report. Bone biopsy is necessary for an accurate diagnosis.

Effect of Neoadjuvant Therapies on Soft Tissue Sarcomas with Tail-like Lesions: A Multicenter Retrospective Study.

Several types of soft tissue sarcomas have peripheral infiltrative growth characteristics called tail-like lesions. The efficacy of neoadjuvant therapy for tumors with tail-like lesions has not been elucidated. From 2012 to 2019, we analyzed 36 patients with soft tissue sarcoma with tail-like lesions treated with neoadjuvant therapy, including chemotherapy, radiotherapy, or both. The effect of neoadjuvant therapy on the tail sign was investigated by analyzing the change in tail-like lesions during neoadjuvant therapy and histological responses. The median length of the tail-like lesion reduced from 29.5 mm at initiation to 19.5 mm after neoadjuvant therapy. The extent of shrinkage in tail-like lesions was related to the histopathological responses in the main part of the tumor. Complete disappearance of the tail-like lesion was observed in 12 patients; however, it was not related to achieving a microscopically negative margin. The oncologic outcomes did not significantly differ between cases with and without the complete disappearance of tail-like lesions. This study indicated that the shrinkage of tail-like lesions did not have a significant effect on complete resection or improvements of clinical outcomes. A more comprehensive evaluation is needed to elaborate on the surgical strategy.

Different patterns of pneumothorax in patients with soft tissue tumors treated with pazopanib: A case series analysis.

To investigate pneumothorax patterns in pazopanib treatment by focusing on the positional relationship between the visceral pleura and metastatic lung tumor, we examined 20 patients with advanced soft tissue tumors who developed lung metastases and underwent pazopanib treatment between 2012 and 2019. Pneumothorax was classified into two types based on the location of the metastatic lesion around the visceral pleural area before pazopanib treatment: subpleural type, within 5 mm from the pleura; and central type, >5 mm from the pleura. We investigated the rates of pneumothorax and the associated risk factors. Five patients experienced pneumothorax (three subpleural and two central types). Cavitation preceded pneumothorax in 83% of patients and led to connection of the cavitated cyst of the metastatic lesion to the chest cavity in the shorter term in patients with the subpleural type. Conversely, a more gradual increase in the cavity size and sudden cyst rupture were observed in the central type. The risk factors for pneumothorax were cavitation after initiating pazopanib and intervention before pazopanib, either ablation or surgery. The location of the metastatic lesions was not a risk factor for the occurrence of pneumothorax. In conclusion, pneumothorax is an adverse event associated with pazopanib treatment. Therefore, attention must be paid to predisposing factors such as the formation of cavitation after pazopanib initiation and previous interventions to the lungs. Moreover, because subpleural pneumothorax tends to occur earlier than the central type, a different time course can be anticipated based on the positional relationships of the metastatic lesions to the visceral pleura.

Myelolipoma mimicking osteosarcoma in the distal femur.

INTRODUCTION AND IMPORTANCE: Periosteal reactions indicate malignant bone tumors, including osteosarcoma; establishing an accurate diagnosis is key to determining the most appropriate treatment strategy. We describe a rare case of myelolipoma in the distal femur metaphysis with massive extraskeletal lesions and periosteal reactions. CASE PRESENTATION: A 25-year-old woman was referred to our hospital to treat a gradually expanding mass around her knee that grew to the size of a baby's head. She had a history of hydrocephalus caused by congenital cytomegalovirus infection and was bedridden for life. Radiography showed a prominent osteoblastic rim and osteolytic lesion with a moth-eaten appearance. Osteosarcoma was suspected due to excessive extraskeletal invasion and periosteal reactions. T1- and T2-weighted magnetic resonance images showed a high-signal-intensity homologous lesion. Biopsy specimens contained adipose and hematopoietic tissues. A myelolipoma was diagnosed. Due to her fragility, surgical intervention was suspended. Two years after diagnosis, the tumor size did not change. CLINICAL DISCUSSION: Myelolipomas are benign tumors that typically arise from the adrenal gland and rarely develop in the extremities. This type of tumor typically does not cause any tumor-related symptoms or endocrine disturbances and has been reported as a type of incidentaloma. To effectively manage myelolipoma patients, differential diagnosis of tumors mimicking malignant bone tumors is important. CONCLUSION: We successfully managed a destructive ectopic myelolipoma in the distal femoral metaphysis, with massive extraskeletal lesions and periosteal reactions. Clinicians should appropriately differentiate myelolipoma from tumors mimicking malignant bone tumors.

Micrometer scale guidance of mesenchymal stem cells to form structurally oriented large-scale tissue engineered cartilage.

Current clinical methods to treat articular cartilage lesions provide temporary relief of the symptoms but fail to permanently restore the damaged tissue. Tissue engineering, using mesenchymal stem cells (MSCs) combined with scaffolds and bioactive factors, is viewed as a promising method for repairing cartilage injuries. However, current tissue engineered constructs display inferior mechanical properties compared to native articular cartilage, which could be attributed to the lack of structural organization of the extracellular matrix (ECM) of these engineered constructs in comparison to the highly oriented structure of articular cartilage ECM. We previously showed that we can guide MSCs undergoing chondrogenesis to align using microscale guidance channels on the surface of a two-dimensional (2-D) collagen scaffold, which resulted in the deposition of aligned ECM within the channels and enhanced mechanical properties of the constructs. In this study, we developed a technique to roll 2-D collagen scaffolds containing MSCs within guidance channels in order to produce a large-scale, three-dimensional (3-D) tissue engineered cartilage constructs with enhanced mechanical properties compared to current constructs. After rolling the MSC-scaffold constructs into a 3-D cylindrical structure, the constructs were cultured for 21days under chondrogenic culture conditions. The microstructure architecture and mechanical properties of the constructs were evaluated using imaging and compressive testing. Histology and immunohistochemistry of the constructs showed extensive glycosaminoglycan (GAG) and collagen type II deposition. Second harmonic generation imaging and Picrosirius red staining indicated alignment of neo-collagen fibers within the guidance channels of the constructs. Mechanical testing indicated that constructs containing the guidance channels displayed enhanced compressive properties compared to control constructs without these channels. In conclusion, using a novel roll-up method, we have developed large scale MSC based tissue-engineered cartilage that shows microscale structural organization and enhanced compressive properties compared to current tissue engineered constructs. STATEMENT OF SIGNIFICANCE: Tissue engineered cartilage constructs made with human mesenchymal stem cells (hMSCs), scaffolds and bioactive factors are a promising solution to treat cartilage defects. A major disadvantage of these constructs is their inferior mechanical properties compared to the native tissue, which is likely due to the lack of structural organization of the extracellular matrix of the engineered constructs. In this study, we developed three-dimensional (3-D) cartilage constructs from rectangular scaffold sheets containing hMSCs in micro-guidance channels and characterized their mechanical properties and metabolic requirements. The work led to a novel roll-up method to embed 2-D microscale structures in 3-D constructs. Further, micro-guidance channels incorporated within the 3-D cartilage constructs led to the production of aligned cell-produced matrix and enhanced mechanical function.

Macrophage-secreted granulin supports pancreatic cancer metastasis by inducing liver fibrosis.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating metastatic disease for which better therapies are urgently needed. Macrophages enhance metastasis in many cancer types; however, the role of macrophages in PDAC liver metastasis remains poorly understood. Here we found that PDAC liver metastasis critically depends on the early recruitment of granulin-secreting inflammatory monocytes to the liver. Mechanistically, we demonstrate that granulin secretion by metastasis-associated macrophages (MAMs) activates resident hepatic stellate cells (hStCs) into myofibroblasts that secrete periostin, resulting in a fibrotic microenvironment that sustains metastatic tumour growth. Disruption of MAM recruitment or genetic depletion of granulin reduced hStC activation and liver metastasis. Interestingly, we found that circulating monocytes and hepatic MAMs in PDAC patients express high levels of granulin. These findings suggest that recruitment of granulin-expressing inflammatory monocytes plays a key role in PDAC metastasis and may serve as a potential therapeutic target for PDAC liver metastasis.

Three Huntington's Disease Specific Mutation-Carrying Human Embryonic Stem Cell Lines Have Stable Number of CAG Repeats upon In Vitro Differentiation into Cardiomyocytes.

Huntington disease (HD; OMIM 143100), a progressive neurodegenerative disorder, is caused by an expanded trinucleotide CAG (polyQ) motif in the HTT gene. Cardiovascular symptoms, often present in early stage HD patients, are, in general, ascribed to dysautonomia. However, cardio-specific expression of polyQ peptides caused pathological response in murine models, suggesting the presence of a nervous system-independent heart phenotype in HD patients. A positive correlation between the CAG repeat size and severity of symptoms observed in HD patients has also been observed in in vitro HD cellular models. Here, we test the suitability of human embryonic stem cell (hESC) lines carrying HD-specific mutation as in vitro models for understanding molecular mechanisms of cardiac pathology seen in HD patients. We have differentiated three HD-hESC lines into cardiomyocytes and investigated CAG stability up to 60 days after starting differentiation. To assess CAG stability in other tissues, the lines were also subjected to in vivo differentiation into teratomas for 10 weeks. Neither directed differentiation into cardiomyocytes in vitro nor in vivo differentiation into teratomas, rich in immature neuronal tissue, led to an increase in the number of CAG repeats. Although the CAG stability might be cell line-dependent, induced pluripotent stem cells generated from patients with larger numbers of CAG repeats could have an advantage as a research tool for understanding cardiac symptoms of HD patients.

A thyroid hormone receptor/KLF9 axis in human hepatocytes and pluripotent stem cells.

Biological processes require close cooperation of multiple transcription factors that integrate different signals. Thyroid hormone receptors (TRs) induce Krüppel-like factor 9 (KLF9) to regulate neurogenesis. Here, we show that triiodothyronine (T3) also works through TR to induce KLF9 in HepG2 liver cells, mouse liver, and mouse and human primary hepatocytes and sought to understand TR/KLF9 network function in the hepatocyte lineage and stem cells. Knockdown experiments reveal that KLF9 regulates hundreds of HepG2 target genes and modulates T3 response. Together, T3 and KLF9 target genes influence pathways implicated in stem cell self-renewal and differentiation, including Notch signaling, and we verify that T3 and KLF9 cooperate to regulate key Notch pathway genes and work independently to regulate others. T3 also induces KLF9 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) and this effect persists during differentiation to definitive endoderm and hiPSC-derived hepatocytes. Microarray analysis reveals that T3 regulates hundreds of hESC and hiPSC target genes that cluster into many of the same pathways implicated in TR and KLF9 regulation in HepG2 cells. KLF9 knockdown confirms that TR and KLF9 cooperate to regulate Notch pathway genes in hESC and hiPSC, albeit in a partly cell-specific manner. Broader analysis of T3 responsive hESC/hiPSC genes suggests that TRs regulate multiple early steps in ESC differentiation. We propose that TRs cooperate with KLF9 to regulate hepatocyte proliferation and differentiation and early stages of organogenesis and that TRs exert widespread and important influences on ESC biology.

Micrometer scale guidance of mesenchymal stem cells to form structurally oriented cartilage extracellular matrix.

Tissue engineering is a possible method for long-term repair of cartilage lesions, but current tissue-engineered cartilage constructs have inferior mechanical properties compared to native cartilage. This problem may be due to the lack of an oriented structure in the constructs at the microscale that is present in the native tissue. In this study, we utilize contact guidance to develop constructs with microscale architecture for improved chondrogenesis and function. Stable channels of varying microscale dimensions were formed in collagen-based and polydimethylsiloxane membranes via a combination of microfabrication and soft-lithography. Human mesenchymal stem cells (MSCs) were selectively seeded in these channels. The chondrogenic potential of MSCs seeded in these channels was investigated by culturing them for 3 weeks under differentiating conditions, and then evaluating the subsequent synthesized tissue for mechanical function and by type II collagen immunohistochemistry. We demonstrate selective seeding of viable MSCs within the channels. MSC aligned and produced mature collagen fibrils along the length of the channel in smaller linear channels of widths 25-100 µm compared to larger linear channels of widths 500-1000 µm. Further, substrates with microchannels that led to cell alignment also led to superior mechanical properties compared to constructs with randomly seeded cells or selectively seeded cells in larger channels. The ultimate stress and modulus of elasticity of constructs with cells seeded in smaller channels increased by as much as fourfolds. We conclude that microscale guidance is useful to produce oriented cartilage structures with improved mechanical properties. These findings can be used to fabricate large clinically useful MSC-cartilage constructs with superior mechanical properties.

Fibronectin is essential for survival but is dispensable for proliferation of hepatocytes in acute liver injury in mice.

UNLABELLED: Acute liver injury causes massive hepatocyte apoptosis and/or fatal liver damage. Fibronectin, an extracellular matrix glycoprotein, is prominently expressed during adult tissue repair. However, the extent of fibronectin dependence on hepatocyte response to acute liver damage remains to be defined. Because identification of hepatic survival factors is critical for successful therapeutic intervention in liver failure, this relationship has been investigated using a fibronectin-deficient mouse model of acute liver injury. Here, we show that lack of fibronectin induces significantly increased hepatocyte apoptosis, which is accompanied by significant down-regulation of the antiapoptotic protein, B-cell lymphoma-extra large (Bcl-xL). Furthermore, fibronectin deficiency leads to a significantly elevated production of hepatocyte growth factor in hepatic stellate cells postinjury, which, in turn, results in an earlier onset and acceleration of hepatocyte regeneration. Primary hepatocytes on fibronectin are protected from reactive oxygen species-induced cellular damage, retaining the expression of Bcl-xL, whereas those on type I collagen are not. This retained expression of Bcl-xL is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. CONCLUSION: We provide evidence that fibronectin-mediated matrix survival signals for hepatocytes are transduced through the PI3K/Bcl-xL-signaling axis in response to injury. This work defines fibronectin as a novel antiapoptotic factor for hepatocytes after acute liver injury, but demonstrates that fibronectin is not essential for subsequent hepatocyte proliferation.

Musculoskeletal diseases--tendon.

INTRODUCTION: Tendons establish specific connections between muscles and the skeleton by transferring contraction forces from skeletal muscle to bone thereby allowing body movement. Tendon physiology and pathology are heavily dependent on mechanical stimuli. Tendon injuries clinically represent a serious and still unresolved problem since damaged tendon tissues heal very slowly and no surgical treatment can restore a damaged tendon to its normal structural integrity and mechanical strength. Understanding how mechanical stimuli regulate tendon tissue homeostasis and regeneration will improve the treatment of adult tendon injuries that still pose a great challenge in today's medicine. SOURCE OF DATA: This review summarizes the current status of tendon treatment and discusses new directions from the point of view of cell-based therapy and regenerative medicine approach. We searched the available literature using PubMed for relevant original articles and reviews. GROWING POINTS: Identification of tendon cell markers has enabled us to study precisely tendon healing and homeostasis. Clinically, tissue engineering for tendon injuries is an emerging technology comprising elements from the fields of cellular source, scaffold materials, growth factors/cytokines and gene delivering systems. AREAS TIMELY FOR DEVELOPING RESEARCH: The clinical settings to establish appropriate microenvironment for injured tendons with the combination of these novel cellular- and molecular-based scaffolds will be critical for the treatment.

Pericellular fibronectin is required for RhoA-dependent responses to cyclic strain in fibroblasts.

To test the hypothesis that the pericellular fibronectin matrix is involved in mechanotransduction, we compared the response of normal and fibronectin-deficient mouse fibroblasts to cyclic substrate strain. Normal fibroblasts seeded on vitronectin in fibronectin-depleted medium deposited their own fibronectin matrix. In cultures exposed to cyclic strain, RhoA was activated, actin-stress fibers became more prominent, MAL/MKL1 shuttled to the nucleus, and mRNA encoding tenascin-C was induced. By contrast, these RhoA-dependent responses to cyclic strain were suppressed in fibronectin knockdown or knockout fibroblasts grown under identical conditions. On vitronectin substrate, fibronectin-deficient cells lacked fibrillar adhesions containing alpha5 integrin. However, when fibronectin-deficient fibroblasts were plated on exogenous fibronectin, their defects in adhesions and mechanotransduction were restored. Studies with fragments indicated that both the RGD-synergy site and the adjacent heparin-binding region of fibronectin were required for full activity in mechanotransduction, but not its ability to self-assemble. In contrast to RhoA-mediated responses, activation of Erk1/2 and PKB/Akt by cyclic strain was not affected in fibronectin-deficient cells. Our results indicate that pericellular fibronectin secreted by normal fibroblasts is a necessary component of the strain-sensing machinery. Supporting this hypothesis, induction of cellular tenascin-C by cyclic strain was suppressed by addition of exogenous tenascin-C, which interferes with fibronectin-mediated cell spreading.

Induction of an epithelial integrin alphavbeta6 in human cytomegalovirus-infected endothelial cells leads to activation of transforming growth factor-beta1 and increased collagen production.

Human cytomegalovirus (CMV) infection is a major cause of morbidity in immunosuppressed individuals, and congenital CMV infection is a leading cause of birth defects in newborns. Infection with pathogenic viral strains alters cell-cell and cell-matrix interactions, affecting extracellular matrix remodeling and endothelial cell migration. The multifunctional cytokine transforming growth factor (TGF)-beta1 regulates cell proliferation, differentiation, and extracellular matrix remodeling. Secreted as a latent protein complex, TGF-beta1 requires activation before binding to receptors that phosphorylate intracellular effectors. TGF-beta1 is activated by integrin alphavbeta6, which is strongly induced in the epithelium by injury and inflammation but has not previously been found in endothelial cells. Here, we report that CMV infection induces integrin alphavbeta6 expression in endothelial cells, leading to activation of TGF-beta1, signaling through its receptor ALK5, and phosphorylation of its intracellular effector Smad3. Infection of endothelial cells was also found to stimulate collagen synthesis through a mechanism dependent on both TGF-beta1 and integrin alphavbeta6. Immunohistochemical analysis showed integrin alphavbeta6 up-regulation in capillaries proximal to foci of CMV infection in lungs, salivary glands, uterine decidua, and injured chorionic villi of the placenta, demonstrating both its induction in endothelium and up-regulation in epithelium in vivo. Our results suggest that activation of TGF-beta1 by integrin alphavbeta6 contributes to pathological changes and may impair endothelial cell functions in tissues that are chronically infected with CMV.

Epidermal hyperplasia and papillomatosis in mice with a keratinocyte-restricted deletion of csk.

The Src family kinases (SFKs) are believed to play critical roles in malignant transformation, as well as in growth, invasion and dissemination of neoplastic tissue. Inhibition of SFK-mediated signal transduction and activation of downstream targets inhibits tumor progression. To determine whether constitutive activity of SFK per se is sufficient to induce tumorigenesis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of the SFK-negative regulator csk (Csk-K5 mice). Even though expression levels of SFKs were lower in C-terminal Src kinase (Csk)-null keratinocytes, activity levels were higher than in control keratinocytes. At the age of 3 months, all Csk-K5 mice displayed signs of chronic inflammation in dermis and epidermal hyperplasia. About 19% of Csk-K5 mice (7 out of 36) developed papillomatous lesions. However, these lesions did not show any signs of neoplastic transformation over the next 8 months. Epidermal hyperplasia and hyperkeratosis in Csk-K5 mice were associated with an increased number of stem cells in the interfollicular epidermis, an increased proliferation of basal keratinocytes and a delayed terminal differentiation of the suprabasal keratinocytes. Our results clearly demonstrate that even though SFK-mediated signaling promotes tumor progression, elevated activity of SFKs in vivo alone is not sufficient to induce neoplastic transformation.

Fibronectin is required for integrin alphavbeta6-mediated activation of latent TGF-beta complexes containing LTBP-1.

Transforming growth factor-betas (TGF-beta) are secreted as latent complexes consisting of the TGF-beta dimer, the TGF-beta propeptide dimer, and the latent TGF-beta binding protein (LTBP). Although the bonds between TGF-beta and its propeptide are cleaved intracellulary, the propeptide associates with TGF-beta by electrostatic interactions, thereby conferring latency to the complex. We reported that a specific sequence of LTBP-1 is required for latent TGF-beta activation by the integrin alphavbeta6. Here we describe a 24 amino acid sequence from the hinge domain required for activation. The LTBP-1 polypeptide rL1N, which includes the hinge, associates with fibronectin in binding assays. We present evidence that fibronectin null cells minimally activate latent TGF-beta and poorly incorporate the active hinge sequence into their matrix. In addition, cells missing the fibronectin receptor alpha5beta1 exhibit defective activation of latent TGF-beta by alphavbeta6 and decreased matrix incorporation. The results indicate specificity for integrin-mediated latent TGF-beta activation that include unique sequences in LTBP-1 and an appropriate matrix molecule.

Integrin alphavbeta3 mediates platelet-derived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin alpha2beta1.

The interplay between the collagen-binding integrin, alpha2beta1, and platelet-derived growth factor (PDGF) receptors in the context of functional interactions with collagen was studied. We expressed either wild-type alpha2beta1 (alpha2beta1A) or alpha2beta1 with a Y783/795F mutation in the cytoplasmic tail of the beta1 subunit (alpha2beta1Amut) in the beta1-null fibroblastic cell line, GD25. GD25 cells lack endogenous expression of the alpha1 and alpha2 integrin subunits and do not adhere to collagen even after transfection with beta1A. Cells expressing alpha2beta1Amut contracted three-dimensional collagen lattices less efficiently than those expressing alpha2beta1A. PDGF-BB significantly stimulated lattice contraction by GD25-alpha2beta1Amut cells. Both cell types responded chemotactically to PDGF-BB. Focal adhesion kinase (FAK) and p130(Cas) were phosphorylated when GD25-alpha2beta1A cells, but not GD25-alpha2beta1Amut cells were seeded on collagen-coated dishes. Subsequent treatment with PDGF-BB further increased phosphorylation of FAK and p130(Cas) only in GD25-alpha2beta1A cells. However, when cultured within collagen lattices, FAK and p130(Cas) phosphorylation were stimulated in both alpha2beta1A- and alpha2beta1Amut-expressing cells but further phosphorylation, in response to subsequent treatment with PDGF-BB, was seen only in GD25-alpha2beta1A cells. We show that the stimulatory effects of PDGF-BB on collagen gel contraction and chemotaxis by GD25-alpha2beta1Amut cells were mediated by the alphavbeta3 integrin. Phosphorylation of p130(Cas), but not FAK, in GD25-alpha2beta1Amut cells seeded in collagen lattices also depended on alphavbeta3. Our results show that PDGF-BB stimulation of fibroblast-collagen interactions is mediated by the alphavbeta3 integrin when beta1 integrin function is impaired.

Antiangiogenic proteins require plasma fibronectin or vitronectin for in vivo activity.

Fragmentation of various extracellular matrix and blood proteins generates antiangiogenic substances that are physiological regulators of angiogenesis. Some of these compounds are in clinical trials as inhibitors of tumor angiogenesis. Anastellin, an antiangiogenic protein fragment derived from fibronectin, was unable to inhibit matrigel plug angiogenesis in mice that lack plasma fibronectin. Anastellin was fully active in mice that are null for vitronectin, which, like fibronectin, is a major adhesion protein in the blood. An antiangiogenic form of antithrombin showed the opposite pattern. The activity of endostatin was impaired in both fibronectin- and vitronectin-deficient mice. These results suggest a shared mechanism of action for antiangiogenic factors derived from extracellular matrix and plasma proteins: these factors form complexes with adhesion proteins in plasma to create an active antiangiogenic substance.