Extracellular matrix structure and nano-mechanics determine megakaryocyte function

Collagen fibrils are the main architectural element in several tissues and bones, and the self-assembly processes involved in collagen fibrillogenesis are of enormous importance to matrix pathology and proper cell development.¹  Type I collagen plays a critical role in the bone marrow niche environment, preventing platelet formation by MKs through the activation of integrin-α2β1 and the Rho-ROCK pathway.^2-4  Changes in mechanical properties are tightly coupled in vivo to changes in matrix composition, crosslinking, and density.⁵  Moreover, different signaling pathways, related to the transduction of mechanical signals into biochemical responses, regulate cell behavior.⁶  However, the question on how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate MK fate, is still open. Therefore, our purpose for the present work was to focus on MK function related to type I collagen organization and biomechanics.

Type I collagen from bovine tendon was modified as previously described⁹  and as reported in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Antibodies and solid-phase binding assay are described in supplemental Methods. MKs were differentiated from human cord blood progenitors as previously described as well as proplatelet and spreading assay, electron and immunofluorescence microscopy, second harmonic generation, and Western blotting analysis.^4,7,8  Human cord blood was collected on informed consent of the parents, with approval from the ethical committee of the Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo Foundation in accordance with the principles of the Declaration of Helsinki. Binding of cells to different collagen preparations was evaluated with 25 × 10³ cells seeded for 1 hour in Stem Span medium (Stem Cell Technologies) in a 96-well plate precoated with 5 μg of each collagen preparation. Cells were preincubated with increasing concentrations of anti-α2 (clone P1E6) and anti-GPVI (Fab 9012.2) antibodies. Adhering cells were washed after 1 hour with PBS and counted by phase-contrast microscopy. Cell adhesion values are expressed relative to control (absence of inhibitor). In some experiments, cells were seeded on 12-mm glass coverslips coated with type I collagen, allowed to adhere for 2 hours in the presence of 20 μg/mL of antibodies, and then stained for actin and CD41. For cell migration experiments, 25 × 10³ cells were seeded in Stem Span medium in the upper well of transwell migration chambers (8 μm; Millipore). Migration toward 100 ng/mL of SDF-1 (PeproTech) and through filters coated with different collagen preparations (5 μg/well) or BSA was measured after 16 hours. Migrated cells were cytospun, fixed, stained with anti-CD41 antibody, and counted. Atomic force microscopy (AFM) force measurements were performed on a MFP-3D bio-atomic force microscope (Asylum Research) using triangular silicon nitride cantilever probes with nominal tip radius of 42 nm and a nominal spring constant approximately 20 pN/m, as determined from thermal calibration. Young moduli of collagens and cells were derived from force versus indentation profile and extracted using a Hertz model¹⁰  with the following formula:

with a tip opening angle of 36°, a tip Poisson ratio (V) of 0.25, a sample Poisson ratio (V) set to 0.5, and a tip modulus (E) of 290 GPa. E was determined by a least squared fit using Igor Pro Version 6.20 data analysis software (WaveMetrics). Cells were fixed with paraformaldehyde 3%, and force curves were taken in spread cells with similar dimension and height. Cells were indented at a site midway between the nucleus and cell margin, and at least 10 force curves were acquired per cell. t test was used to analyze data, with a significant difference set at P < .05. Data are presented as mean ± SD.

The relationships between MK and bone marrow components are key factors in platelet formation. Importantly, changes in mechanical properties are pervasive in vivo during pathologic processes, such as fibrosis or tumorigenesis.¹¹  Based on insight from the literature as well as from our prior studies,⁷  we aimed to interrogate the synergy between biochemical and biophysical processes related to MK development within the bone marrow environment. To pursue this goal, we used a chemically modified type I collagen by N-acetylation of lysine side chains, which blocks the formation of banded fibrils in neutral conditions (Figure 1A).⁹ 

AFM and second harmonic generation imaging, a noninvasive optical method that exploits nonlinear light scattering from fibrillar collagen, showed that dehydrated native type I collagen formed a layer of banded fibrils with D-periodicity, whereas the N-acetylated type I collagen was characterized by microfibrillar structure in the early stage of aggregation (Figure 1B). Consistently, AFM nano-topography in fluids showed that, starting from 2 hours of incubation, coatings of native type I collagen, and not N-acetylated collagen, were characterized by the presence of fibrils, on a layer of unorganized material, homogeneously distributed with a length ranging from 1 μm up to several microns (Figure 1C). Nano-indentation studies with AFM demonstrated that type I collagen had a heterogeneous distribution of elastic moduli that derived from points picked on fibrils and points picked on the underlying material. In particular, fibrils in fluids presented an elasticity > 0.15 GPa, whereas material in early stage of self-aggregation had values in the order of 5-75 MPa. On the contrary, homogeneous values in the order of < 75 MPa were found during the analysis of N-acetylated collagen Young moduli.

MKs express 2 main receptors for collagen: integrin-α2β1 and glycoprotein VI.¹²  Using function-blocking antibodies, a prominent role for integrin-α2β1, and not GPVI, emerged in the early phase of MK adhesion on collagen, as demonstrated by a significant reduction in cell adhesion and spreading (Figure 1D). Importantly, binding to collagen receptors was not affected by the lack of self-aggregation of N-acetylated type I collagen because no significant differences were found in MK adhesion in the presence of increasing concentrations of anti-α2 or anti-GPVI antibodies (Figure 1E).

To analyze whether alterations in type I collagen nano-mechanical properties could affect MK function, human MKs were allowed to adhere on native and N-acetylated collagens for 2 and 16 hours. After 2 hours, ∼ 20% of cells were spread on both collagens (Figure 2B); however, prolonging incubation to 16 hours, MKs adherent on N-acetylated collagen started to lose cell contractility, returned round and extended proplatelets as control MKs cultured in suspension (Figure 2A-C). On the contrary, on the native type I collagen, MKs maintained cell contractility, remained spread over the entire time of incubation, and did not extend proplatelets (Figure 2A-C). Overall, these data demonstrated, for the first time, that decreasing type I collagen stiffness along with a nonorganized fibrillar structure completely reverted type I collagen-dependent functions of human MKs. Consistently, only stiffer native type I collagen, and not the N-acetylated one, was able to refrain MK migration (Figure 2D). These data demonstrated that type I collagen fibrils provide structural cues for MK adhesion and migration.¹³  To further demonstrate that MKs anchored more strongly to stiffer type I collagen than to the softer N-acetylated one, we took advantage of our recent data on fibronectin (FN) as a mediator of MK adhesion to type I collagen.⁸  Binding of FN to native and N-acetylated collagen was preserved as verified in solid-phase assays (supplemental Figure 1). Nevertheless, we demonstrated that only adhesion on fibrillar type I collagen led to the assembly of FITC-labeled FN in insoluble matrix on MK surface (Figure 2E) as well as to stable relocation to the plasma membrane of endogenous FN (Figure 2F). Moreover, to demonstrate the feedback of local matrix stiffness on the MK state, Young moduli values of fixed spread MKs on native and N-acetylated type I collagens were measured. Heterogeneity in native type I collagen stiffness was reflected on membrane of adhering MKs as values of elasticity varied from 10.35-183.62 KPa (mean, 41.86 KPa), whereas more homogeneous distribution of values, from 12.78-59.08 KPa (mean, 24.71 KPa), were detected in MKs adherent on N-acetylated type I collagen (Figure 2G). Different kinases and GTPases regulate MK function in response to collagen, but their activation is not completely unraveled. An α2β1-dependent pathway involving Rho-ROCK-MLC has been demonstrated to negatively regulate proplatelet release,^2,3  whereas Src and Syk phosphorylation, downstream GPVI activation, were shown to be involved in α2bβ3-dependent migration of MK on fibronectin.¹⁴  In our experiments, levels of guanosine triphosphate-bound RhoA resulted comparable on MK adhesion to either type I collagen preparations for 16 hours demonstrating that engagement of integrin-α2β1had occurred. However, an evident reduction of myosin light chain (MLC-2) phosphorylation was observed on MK adhesion on N-acetylated type I collagen with respect to the native one (Figure 2H). Interestingly, engagement of GPVI was shown by Src and Syk phosphorylation, and no differences were observed on adhesion on either collagen preparations (Figure 2H). Overall, our results demonstrated that engagement of integrin-α2β1 is not sufficient to achieve the level of MLC-2 phosphorylation necessary to the inhibition of proplatelet normally exerted by fibrillar type I collagen. Further, GPVI activation in MKs seemed not to be affected by collagen structure and nano-mechanics. Therefore, other, yet unknown, receptors may cooperate with α2β1 to regulate MK function on type I collagen in a structure-dependent manner.

In conclusion, our results provide novel insight into the role of nano-mechanics of a bone marrow matrix-environment related to MK biology, leading to new concepts in understanding how MKs sense the stiffness and spatial patterning of their environment to modulate their function and fate. The identification of mechano-sensing receptors could be a novel approach to study MK behavior that deserves further investigations. Modification of matrix structure within bone marrow in a diseased state, such as myelofibrosis, may represent an important example of aberrant megakaryopoiesis dependent on alteration of bone marrow matrix nano-mechanics.

The authors thank Martine Jandrot-Perrus (Inserm, U698, Hôpital Bichat, Paris, France) for the anti-GPVI antibody, Luigi De Marco (CRO, Aviano, Italy) for helpful discussion, Livia Visai (University of Pavia, Pavia, Italy) for the human fibronectin, Cesare Perotti (Istituto di Ricovero e Cura a Carattere Scientifico San Matteo Foundation, Pavia, Italy) for human cord blood supply, and Luca Monti (University of Pavia, Pavia, Italy) for helping with Western blotting.

This work was supported by the Cariplo Foundation (2006.0596/10.8485), Regione Lombardia (Project SAL-45 and ASTIL 16783; SAL-13, Almamater Foundation, Pavia; A.B.), National Institutes of Health (P41 EB002520; Tissue Engineering Resource Center, D.K.), and Tufts University (faculty startup funds, C.S.).

National Institutes of Health

Contribution: A.M. performed experiments, analyzed data, and wrote the paper; C.G., I.P., E.S., R.T., and M.R. performed experiments; D.K., M.E.T., and C.S. analyzed data; and A.B. designed experiments, analyzed data, wrote the paper, and provided overall direction.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Alessandra Balduini, Biotechnology Laboratories, Department of Biochemistry, Istituto di Ricovero e Cura a Carattere Scientifico San Matteo Foundation, University of Pavia, via Bassi 21, 27100 Pavia, Italy; e-mail: alessandra.balduini@unipv.it.