Mechanotransduction of Low Intensity Vibration in Human Bone Marrow Mesenchymal Stem Cells and Macrophages

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URL: https://ir.stonybrook.edu/xmlui/handle/11401/76996

Abstract

Human bone marrow mesenchymal stem cells in bone marrow niches are sensitive to the low intensity vibration. The question of what specific mechanical signals (i.e. fluid shear, frequency, or acceleration) cells sense requires the understanding of the underlying pathways. The cellular and molecular responses to LIV in vivo require fundamental investigation of human MSCs and macrophages in bone marrow niches. We aim to elucidate mechanotransduction pathways of LIV in hBMSCs including macrophages and their functions in bone marrow niches. Understanding mechanotransduction pathways can provide the successful use of LIV in controls of regulatory bone pathways in vivo.

We first hypothesized that the physical interaction of cellular and nuclear structures could involve in the mechanotransduction. To test the physical structures based mechanotransduction, we vibrated hBMSC cells under horizontal and vertical directions. Signal combinations of LIV (reflecting a range of fluid shear, i.e. the highest fluid shear at 30Hz-1g and the lowest fluid shear at 100Hz-0.15g by horizontal LIV) were used during cell proliferation and differentiation. The standard assay of MTS monitored cell proliferation and the alizarin red s including oil red o assays were used to monitor cell osteoblastogenic- and adipogenic-differentiations. Flow cytometric analysis of the %gated positive cells confirmed the data from these assays and the gene expression. Gene expression levels (in fold changes) with the GAPDH housekeeping gene were analyzed by the real-time PCR. The cluster and enrichment analysis validated the relation of gene expressions in the proposed mechanotransduction pathway. Based on cellular structures, the mechanosensitive system could derive from the outermost cell membrane (n-cadherins), inside cells (cytoskeletal f-actins) and the nucleoskeletons (NuAnCE-LiNC) anchoring throughout the nuclear envelope (nesprins) across the inner nuclear membrane (sun proteins) to the inner nuclear basement membranes (laminins and chromatins). The mechanotransduction of the physical connections between cellular and nuclear structures provided the potential pathway from the outermost cell membrane to inside cells across the nuclear double membranes as ncadherins – cytoskeletal f-actins – nesprins.

We identified the mechanical signals that hBMSCs could sense. The frequency of LIV influenced the proliferation while the direction had strong effects on the osteoblastogenic differentiation of hBMSCs. Gene profiles showed that the physical interactions of cellular and nuclear structures involved in the mechanotransduction. During LIV, cells and their neighboring cells were physical interacting with one another and formed mature adherent junctions (CDH11). The connections of the mature adherent junctions with cytoskeletons were dynamic. The dynamic changes in the cytoskeletal orientations (ACTn1) potentially maintained cellular integrity. The cytoskeletal orientation was then anchoring the nuclear skeleton nesprins (Syne2). The CDH11-ACTn1-Syne2 pathway was the mechanotransduction of LIV in hBMSCs.

Secondly, we hypothesized that LIV could change cytoskeletal orientations and that the structural changes could affect the cellular mechanics. We used two-photon confocal microscopy to visualize cytoskeletal f-actins and validated specific physical interactions of cellular structures based mechanotransduction pathway. Atomic force microscope (with liquid mode) estimated the relative mechanical modulus that changed with the structural changes and remodeling. If cellular mechanical properties were different in LIV protocols, the relative cellular mechanics could provide information on the mechanical sensing mechanisms.

From two-photon images, we found that the cytoskeletal orientation anchoring nucleoskeletons. The changes in cytoskeletal structures driven changes in the mechanical modulus were measured by atomic force microscopy. We found that the cytoskeletal orientation of horizontal LIV was better than that of the vertical LIV. Correspondingly, the mechanical modulus of hLIV was higher than the moduli of vLIV. The cytoskeletal knockdown provided a confirmation of the CDH11-ACTn1-Syne2 mechanotransduction pathway. Without cytoskeletons, we found that the ACTn1-Syne2 was depleted, which confirmed the existing CDH11-ACTn1-Syne2 pathway.

The third hypothesis was to understand biological and molecular functions of LIV driven cytoskeletal orientation. We hypothesized that the cytoskeletal orientation could control differentiation fates of human stem cells. The osteoblastogenic and adipogenic differentiations of hBMSCs and hAMSCs under LIV were investigated with and without an induction of the differentiation media.

Our data supported that the cytoskeletal orientation promoted the osteoblastogenic differentiation but suppressed the adipogenic differentiation of both multilineage human bone marrow and adipose-derived MSCs. These data for the first time showed the LIV mechanisms in controlling the differentiation fates. Understanding LIV promoting the osteoblastogenesis and suppressing the adipogenesis provided strategies towards non-pharmacological programming multipotent stem cells towards preferential lineages.

The fourth hypothesis was to understand dynamic bone homeostasis and healing starting in the bone marrow niche. To understand biological mechanisms of LIV on bone marrow niches’ cells, we investigated the effect of hLIV on macrophages. Understanding what drives macrophages toward the pro-healing phenotypes and linking pro-healing expression with hBMSCs could suggest the bone-healing mechanisms in vivo. Perhaps, LIV induced bone formation in balancing with bone resorption via IL-10 during bone homeostasis. We hypothesized that hLIV induced pro-healing macrophages and upregulated VEGF angiogenesis. The upregulated VEGF increased the osteoblastogenic differentiation of hBMSCs and bone survivals. The relation of pro-healing macrophages and hBMSCs provided fundamental understandings of bone homeostasis and healing in bone marrow niches.

We found that macrophages switched their phenotypes from the inflammatory to prohealing phenotypes by hLIV. The anti-inflammatory cytokine interleukin-10 (IL-10) was increased as if the hLIV induced pro-healing macrophages. Activated IL-10 in the pro-healing macrophages promoted upregulated VEGF and TGFβ. The VEGF expression levels of hLIV were upregulated, supporting angiogenesis potentials of macrophages under hLIV. The upregulated TGFβ was maintained in all hLIV conditions as if macrophages maintained the ability of bone homeostasis. The relation of upregulated VEGF in macrophages with increasing hBMSC-driven osteoblastogenesis has confirmed the bone regeneration starting in the bone marrow niches.

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Cited By (1)

Year	Entry
2015	Pongkitwitoon S. Mechanotransduction of Low Intensity Vibration in Human Bone Marrow Mesenchymal Stem Cells and Macrophages [PhD thesis]. Stony Brook, NY: Stony Brook University; May 2015.