Jan-Niklas Schulz

Gray platelet syndrome and defective thrombo-inflammation in Nbeal2-deficient mice

Platelets are anuclear organelle-rich cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity. The major platelet organelles, α-granules, release proteins that participate in thrombus formation and hemostasis. Proteins stored in α-granules are also thought to play a role in inflammation and wound healing, but their functional significance in vivo is unknown. Mutations in NBEAL2 have been linked to gray platelet syndrome (GPS), a rare bleeding disorder characterized by macrothrombocytopenia, with platelets lacking α-granules. Here we show that Nbeal2-knockout mice display the characteristics of human GPS, with defective α-granule biogenesis in MKs and their absence from platelets. Nbeal2 deficiency did not affect MK differentiation and proplatelet formation in vitro or platelet life span in vivo. Nbeal2-deficient platelets displayed impaired adhesion, aggregation, and coagulant activity ex vivo that translated into defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction following focal cerebral ischemia. In a model of excisional skin wound repair, Nbeal2-deficient mice exhibited impaired development of functional granulation tissue due to severely reduced differentiation of myofibroblasts in the absence of α-granule secretion. This study demonstrates that platelet α-granule constituents are critically required not only for hemostasis but also thrombosis, acute thrombo-inflammatory disease states, and tissue reconstitution after injury.

Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure

Background: What is the biological function of COMP in skin ECM? Results: COMP binds collagens XII and XIV that associate with collagen I fibrils. All three proteins localize to anchoring plaques. Conclusion: COMP acts as an adapter in ECM of healthy skin, organizing the dermal collagen network. Significance: COMP organizes collagen I fibrils into a suprastructure that may contribute to stabilizing cohesion between the upper dermis and the basement membrane zone. The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

Enhanced deposition of cartilage oligomeric matrix protein is a common feature in fibrotic skin pathologies

Skin fibrosis is characterized by activated fibroblasts and an altered architecture of the extracellular matrix. Excessive deposition of extracellular matrix proteins and altered cytokine levels in the dermal collagen matrix are common to several pathological situations such as localized scleroderma and systemic sclerosis, keloids, dermatosclerosis associated with venous ulcers and the fibroproliferative tissue surrounding invasively growing tumors. Which factors contribute to altered organization of dermal collagen matrix in skin fibrosis is not well understood. We recently demonstrated that cartilage oligomeric matrix protein (COMP) functions as organizer of the dermal collagen I network in healthy human skin (Agarwal et al., 2012). Here we show that COMP deposition is enhanced in the dermis in various fibrotic conditions. COMP levels were significantly increased in fibrotic lesions derived from patients with localized scleroderma, in wound tissue and exudates of patients with venous leg ulcers and in the fibrotic stroma of biopsies from patients with basal cell carcinoma. We postulate enhanced deposition of COMP as one of the common factors altering the supramolecular architecture of collagen matrix in fibrotic skin pathologies. Interestingly, COMP remained nearly undetectable in normally healing wounds where myofibroblasts transiently accumulate in the granulation tissue. We conclude that COMP expression is restricted to a fibroblast differentiation state not identical to myofibroblasts which is induced by TGFβ and biomechanical forces.

Reduced Granulation Tissue and Wound Strength in the Absence of α11β1 Integrin

Previous wound healing studies have failed to define a role for either α1β1 or α2β1 integrin in fibroblast-mediated wound contraction, suggesting the involvement of another collagen receptor in this process. Our previous work demonstrated that the integrin subunit α11 is highly induced during wound healing both at the mRNA and protein level, prompting us to investigate and dissect the role of the integrin α11β1 during this process. Therefore, we used mice with a global ablation of either α2 or α11 or both integrin subunits and investigated the repair of excisional wounds. Analyses of wounds demonstrated that α11β1 deficiency results in reduced granulation tissue formation and impaired wound contraction, independently of the presence of α2β1. Our combined in vivo and in vitro data further demonstrate that dermal fibroblasts lacking α11β1 are unable to efficiently convert to myofibroblasts, resulting in scar tissue with compromised tensile strength. Moreover, we suggest that the reduced stability of the scar is a consequence of poor collagen remodeling in α11−/− wounds associated with defective transforming growth factor-β–dependent JNK signaling.

COMP-assisted collagen secretion--a novel intracellular function required for fibrosis.

ABSTRACT Cartilage oligomeric matrix protein (COMP) is an abundant component in the extracellular matrix (ECM) of load-bearing tissues such as tendons and cartilage. It provides adaptor functions by bridging different ECM structures. We have previously shown that COMP is also a constitutive component of healthy human skin and is strongly induced in fibrosis. It binds directly and with high affinity to collagen I and to collagen XII that decorates the surface of collagen I fibrils. We demonstrate here that lack of COMP–collagen interaction in the extracellular space leads to changes in collagen fibril morphology and density, resulting in altered skin biomechanical properties. Surprisingly, COMP also fulfills an important intracellular function in assisting efficient secretion of collagens, which were retained in the endoplasmic reticulum of COMP-null fibroblasts. Accordingly, COMP-null mice showed severely attenuated fibrotic responses in skin. Collagen secretion was fully restored by introducing wild-type COMP. Hence, our work unravels a new, non-structural and intracellular function of the ECM protein COMP in controlling collagen secretion. Highlighted Article: In addition to its known function in organizing extracellular collagen fibrils, COMP is identified as assisting in secretion of collagen. COMP-null mice display significantly attenuated skin fibrosis.

The dermatan sulfate proteoglycan decorin modulates α2β1 integrin and the vimentin intermediate filament system during collagen synthesis.

Decorin, a small leucine-rich proteoglycan harboring a dermatan sulfate chain at its N-terminus, is involved in regulating matrix organization and cell signaling. Loss of the dermatan sulfate of decorin leads to an Ehlers-Danlos syndrome characterized by delayed wound healing. Decorin-null (Dcn−/−) mice display a phenotype similar to that of EDS patients. The fibrillar collagen phenotype of Dcn−/− mice could be rescued in vitro by decorin but not with decorin lacking the glycosaminoglycan chain. We utilized a 3D cell culture model to investigate the impact of the altered extracellular matrix on Dcn−/− fibroblasts. Using 2D gel electrophoresis followed by mass spectrometry, we identified vimentin as one of the proteins that was differentially upregulated by the presence of decorin. We discovered that a decorin-deficient matrix leads to abnormal nuclear morphology in the Dcn−/− fibroblasts. This phenotype could be rescued by the decorin proteoglycan but less efficiently by the decorin protein core. Decorin treatment led to a significant reduction of the α2β1 integrin at day 6 in Dcn−/− fibroblasts, whereas the protein core had no effect on β1. Interestingly, only the decorin core induced mRNA synthesis, phosphorylation and de novo synthesis of vimentin indicating that the proteoglycan decorin in the extracellular matrix stabilizes the vimentin intermediate filament system. We could support these results in vivo, because the dermis of wild-type mice have more vimentin and less β1 integrin compared to Dcn−/−. Furthermore, the α2β1 null fibroblasts also showed a reduced amount of vimentin compared to wild-type. These data show for the first time that decorin has an impact on the biology of α2β1 integrin and the vimentin intermediate filament system. Moreover, our findings provide a mechanistic explanation for the reported defects in wound healing associated with the Dcn−/− phenotype.

Dwarfism in Mice Lacking Collagen-binding Integrins α2β1 and α11β1 Is Caused by Severely Diminished IGF-1 Levels

Background: Mice lacking integrins α2β1 and α11β1 are dwarfs. Results: Bones are shorter and less mineralized in absence of osteoblast-specific defects. Severely reduced IGF-1, GH, and GHRH levels result in proportional dwarfism. Conclusion: Integrins α2β1 and α11β1 crucially regulate IGF-1 levels. Significance: We present a novel concept for the role of integrins in growth control, thereby coupling ECM signaling to endocrine homeostasis. Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis.

Controlling the Balance of Fibroblast Proliferation and Differentiation: Impact of Thy-1

Fibroblasts provide matrix and regulatory mediators to the microenvironment and thereby contribute to maintenance of tissue homeostasis, wound healing, and tumor progression. In the present study, we addressed the functional relevance of Thy-1 for fibroblast functions in vitro and in vivo. Using fibroblasts from Thy-1(-/-) and wild-type mice, recombinant expression of Thy-1, and analysis of the interaction of fibroblasts with immobilized Thy-1, we showed that Thy-1 has a crucial role in the control of cell growth by suppressing proliferation and promoting apoptosis and differentiation of dermal fibroblasts. Function-blocking studies revealed that Thy-1 mediates the control of apoptosis and proliferation via modulation of β3 integrin function. Interestingly, Thy-1-mediated growth control appears to be a more general mechanism because it also regulates growth of tumor cells of different origin dependent on their β3 integrin expression. In summary, our findings point to an important role of Thy-1 in controlling the balance between proliferation and differentiation in dermal fibroblasts.

New developments on skin fibrosis - Essential signals emanating from the extracellular matrix for the control of myofibroblasts

Many different diseases are associated with fibrosis of the skin. The clinical symptoms can vary considerably with a broad range from isolated small areas to the involvement of the entire integument. Fibrosis is triggered by a multitude of different stimuli leading to activation of the immune and vascular system that then initiate fibroblast activation and formation of matrix depositing and remodeling myofibroblasts. Ultimately, myofibroblasts deposit excessive amounts of extracellular matrix with a pathological architecture and alterations in growth factor binding and biomechanical properties, which culminates in skin hardening and loss of mobility. Treatment depends certainly on the specific type and cause of the disease, for the autoimmune driven localized and systemic scleroderma therapeutic options are still limited, but recent research has pointed out diverse molecular targets and mechanisms that can be exploited for the development of novel antifibrotic therapy.