Extracellular matrix components

One of the most abundant components of the bone marrow space, besides cells, is a variety of extracellular matrix  components. On selective binding, this environment, in combination with soluble cytokines, regulates haemopoietic progenitor proliferation and differentiation. Haemopietic progenitors develop as adherent cells in contact with extracellular matrix components in the bone marrow until they are released as non-adherent cells into the circulating blood.

Our lab has pursued for long time the hypothesis that the interaction of megakaryocytes with bone marrow extracellular matrix components contributes to the regulation of megakaryocyte function. We have demonstrated that some collagen type can support proplatelet formation, while type I collagen is the only extracellular matrix environment that inhibit this process. Our recent evidence indicates that these differences may be ascribed to peculiar structural properties of the collagens, as well as to differences in receptor engagement. By Atomic Force Microscopy we have documented that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human megakaryocytes through activation of the Rho-ROCK pathway and MLC-2 phosphorylation.

Figure 1: Mechanism model of Mk adhesion to type I collagen in osteoblastic niche environment. Fibronectin assembly is stabilized by FXIII-A activity and reinforces Mk adhesion and spreading on type I collagen. These interactions provide possible mechanisms of sustained inhibition of PPF and platelet shedding within the osteoblastic niche.

Figure 1: Mechanism model of Mk adhesion to type I collagen in osteoblastic niche environment. Fibronectin assembly is stabilized by FXIII-A activity and reinforces Mk adhesion and spreading on type I collagen. These interactions provide possible mechanisms of sustained inhibition of PPF and platelet shedding within the osteoblastic niche.

Recently, we have extended this study in vivo and we have demonstrated that, among bone marrow extracellular matrix components, fibronectin, type IV collagen and laminin are the most abundant around bone marrow sinusoids and constitute a peri-cellular matrix surrounding megakaryocytes. Most importantly, we have shown, for the first time, that megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of megakaryocyte development and bone marrow extracellular matrix homeostasis both in vitro and in vivo. Overall, we have deciphered the extracellular matrix component composition of the megakaryocyte environment and demonstrated that these cells express basement membrane proteins in close proximity to sinusoidal endothelial cells. Importantly, we also showed that extracellular matrix components differently modulate megakaryocyte development in vitro, reflecting the importance of their spatial localization and cell interactions in vivo. Finally, we demonstrated that the production of endogenous extracellular matrix components is not related to the physiological production of platelets in vivo but significantly boosted concomitantly to the regeneration of bone marrow environment following myelosuppression.

Figure 2:  Bone marrow extracellular matrix distribution at endosteal and vascular districts. A) Immunofluorescence analysis of ECM component distribution at endosteal surface and medullary cavity of mouse femur. Images in the endosteal “niche” were acquired at the interface between diaphyseal bone and bone marrow cells. A 20x/0.50 Olympus UPlanF1 objective was used. Scale Bar=100 μm. Hoechst 33258 was used to stain nuclei (blue). B) Confocal microscopy analysis of ex vivo Mk-ECM interaction within bone marrow demonstrated that Mk (CD41+, green) were surrounded by a peri-cellular matrix positive for fibronectin, type IV collagen and laminin (red). Confocal microscopy was performed by a TCS SP2 confocal laser scanning microscope (Leica, Heidelberg, Germany) equipped with a 63× oil-immersion objective. Scale bar=20 μm. Hoechst 33258 was used to stain nuclei (blue).

Figure 2:  Bone marrow extracellular matrix distribution at endosteal and vascular districts. A) Immunofluorescence analysis of ECM component distribution at endosteal surface and medullary cavity of mouse femur. Images in the endosteal “niche” were acquired at the interface between diaphyseal bone and bone marrow cells. A 20x/0.50 Olympus UPlanF1 objective was used. Scale Bar=100 μm. Hoechst 33258 was used to stain nuclei (blue). B) Confocal microscopy analysis of ex vivo Mk-ECM interaction within bone marrow demonstrated that Mk (CD41+, green) were surrounded by a peri-cellular matrix positive for fibronectin, type IV collagen and laminin (red). Confocal microscopy was performed by a TCS SP2 confocal laser scanning microscope (Leica, Heidelberg, Germany) equipped with a 63× oil-immersion objective. Scale bar=20 μm. Hoechst 33258 was used to stain nuclei (blue).

Figure 3:  Recovery of bone marrow niche after myelosuppression triggers the increase of ECM component synthesis by Mks in vivo. A) Schematic representation of the strategy adopted for Mk sorting in platelets depleted mice and 5-FU treated mice. Mk in mice injected with 4μg of anti-GPIbα were sorted between at day 2.5 of treatment and just before the recovery of blood peripheral platelet count. In 5-FU treated mice, Mk were sorted at day 10 of treatment in juxtaposition of bone marrow and pheripheral blood count recovery. Right panel shows a representative hematoxylin & eosin staining of sorted Mks. Scale bar = 10 μm. B) RT-PCR of laminin, type IV collagen chains and fibronectin in Mks treated with PBS (Saline) or anti GPIbα. *p value < 0.05. C) RT-PCR oflaminin, type IV collagen chains and fibronectin in Mks treated with PBS (Saline) or 5-FU. *p value 0.05, **p value 0.01. D) Western blotting analysis of ECM components level in Mks sorted from bone marrow cells after 60 hours of anti GPIbα antibody injection and PBS as control. E) Western blotting analysis of ECM components level in Mks sorted from bone marrow cells of mice myelosuppressed with 5-fluorouracil or PBS as control. β-Actin was revealed to demonstrate equal protein loading. The images are representative of three independent experiments

Figure 3:  Recovery of bone marrow niche after myelosuppression triggers the increase of ECM component synthesis by Mks in vivo. A) Schematic representation of the strategy adopted for Mk sorting in platelets depleted mice and 5-FU treated mice. Mk in mice injected with 4μg of anti-GPIbα were sorted between at day 2.5 of treatment and just before the recovery of blood peripheral platelet count. In 5-FU treated mice, Mk were sorted at day 10 of treatment in juxtaposition of bone marrow and pheripheral blood count recovery. Right panel shows a representative hematoxylin & eosin staining of sorted Mks. Scale bar = 10 μm. B) RT-PCR of laminin, type IV collagen chains and fibronectin in Mks treated with PBS (Saline) or anti GPIbα. *p value < 0.05. C) RT-PCR oflaminin, type IV collagen chains and fibronectin in Mks treated with PBS (Saline) or 5-FU. *p value 0.05, **p value 0.01. D) Western blotting analysis of ECM components level in Mks sorted from bone marrow cells after 60 hours of anti GPIbα antibody injection and PBS as control. E) Western blotting analysis of ECM components level in Mks sorted from bone marrow cells of mice myelosuppressed with 5-fluorouracil or PBS as control. β-Actin was revealed to demonstrate equal protein loading. The images are representative of three independent experiments

Extracellular matrix component Receptor 

Cells use a wide spectrum of proteins and mechanisms to recognize their environment. Evidence demonstrates that extracellular matrix components receptors can be used by Mks to control the site of platelet formation and release. Whether and how extracellular matrix component receptor activity is regulated during thrombopoiesis in vivo is not known. To progress towards a better understanding of these critical mechanisms, significantly improved knowledge of the physical, cellular and biochemical interactions in the bone marrow environment is needed. Integrins are the major human receptors for cell adhesion on extracellular matrix components, however a variety of other interaction mechanisms are possible. Besides integrin alpha2beta1 and GPVI, expression and function of other collagen receptors on human megakaryocytes are unknown. Discoidin domain receptors (DDR1 and DDR2) are tyrosine-kinase collagen receptors that are stimulated by fibrillar and basement membrane collagens and mediate cell adhesion and migration in different tissues. We recently discovered that DDR1 is expressed by both human megakaryocytes and platelets. DDR1 is activated upon megakaryocyte adhesion on fibrillar type I collagen and regulates megakaryocyte Syk-mediated migration through activation of the tyrosine phosphatase SHP1. Altogether, these data point out that DDR1 may represent an important new regulator of megakaryocyte function. 

Figure 4: Human MKs express and synthesize DDR1 tyrosine kinase. A, total cellular RNA was extracted from MKs and fibroblasts (Fb) as positive control. alpha2-microglobulin was used as housekeeping gene. NTC indicates “no template” controls in the reverse transcriptase and PCR steps. RT-PCR products were loaded in duplicates for each cell type. B, MK and fibroblast lysates were subjected to Western blot analysis using an anti-DDR1 antibody. The anti-DDR1 blocking peptide (B.P.) was used to confirm the specificity of the antibody. Actin was probed to show equal loading. C, DDR1 expression was demonstrated in peripheral blood platelet lysate (Plt) by Western blot. Shown here are representative Western blots out of three independent experiments. D, MKs were cytospun on polylysine-coated glass coverslips, fixed, and stained with an anti-DDR1 antibody (red) and an anti-CD61 antibody (green). The graphs report the intensity of the fluorescence signal along the x axis for each fluorochrome on the optical section. Scale bars are 25 mm. Nuclei were counterstained with Hoechst 33288 (blue).

Figure 4: Human MKs express and synthesize DDR1 tyrosine kinase. A, total cellular RNA was extracted from MKs and fibroblasts (Fb) as positive control. alpha2-microglobulin was used as housekeeping gene. NTC indicates “no template” controls in the reverse transcriptase and PCR steps. RT-PCR products were loaded in duplicates for each cell type. B, MK and fibroblast lysates were subjected to Western blot analysis using an anti-DDR1 antibody. The anti-DDR1 blocking peptide (B.P.) was used to confirm the specificity of the antibody. Actin was probed to show equal loading. C, DDR1 expression was demonstrated in peripheral blood platelet lysate (Plt) by Western blot. Shown here are representative Western blots out of three independent experiments. D, MKs were cytospun on polylysine-coated glass coverslips, fixed, and stained with an anti-DDR1 antibody (red) and an anti-CD61 antibody (green). The graphs report the intensity of the fluorescence signal along the x axis for each fluorochrome on the optical section. Scale bars are 25 mm. Nuclei were counterstained with Hoechst 33288 (blue).