Biomechanical forces and force-triggered drug delivery in tumor neovascularization.

Tumor angiogenesis is one of the typical hallmarks of tumor occurrence and development, and tumor neovascularization also exhibits distinct characteristics from normal blood vessels. As the number of cells and matrix inside the tumor increases, the biomechanical force is enhanced, specifically manifested as solid stress, fluid stress, stiffness, and topology. This mechanical microenvironment also provides shelter for tumors and intensifies angiogenesis, providing oxygen and nutritional support for tumor progression. During tumor development, the biomechanical microenvironment also emerges, which in turn feeds back to regulate the tumor progression, including tumor angiogenesis, and biochemical and biomechanical signals can regulate tumor angiogenesis. Blood vessels possess inherent sensitivity to mechanical stimuli, but compared to the extensive research on biochemical signal regulation, the study of the regulation of tumor neovascularization by biomechanical signals remains relatively scarce. Biomechanical forces can affect the phenotypic characteristics and mechanical signaling pathways of tumor blood vessels, directly regulating angiogenesis. Meanwhile, they can indirectly regulate tumor angiogenesis by causing an imbalance in angiogenesis signals and affecting stromal cell function. Understanding the regulatory mechanism of biomechanical forces in tumor angiogenesis is beneficial for better identifying and even taming the mechanical forces involved in angiogenesis, providing new therapeutic targets for tumor vascular normalization. Therefore, we summarized the composition of biomechanical forces and their direct or indirect regulation of tumor neovascularization. In addition, this review discussed the use of biomechanical forces in combination with anti-angiogenic therapies for the treatment of tumors, and biomechanical forces triggered delivery systems.

Similar but distinct: The impact of biomechanical forces and culture age on the production, cargo loading, and biological efficacy of human megakaryocytic extracellular vesicles for applications in cell and gene therapies.

Megakaryocytic extracellular vesicles (MkEVs) promote the growth and megakaryopoiesis of hematopoietic stem and progenitor cells (HSPCs) largely through endogenous miR-486-5p and miR-22-3p cargo. Here, we examine the impact of biomechanical force and culture age/differentiation on the formation, properties, and biological efficacy of MkEVs. We applied biomechanical force to Mks using two methods: shake flask cultures and a syringe pump system. Force increased MkEV production in a magnitude-dependent manner, with similar trends emerging regardless of whether flow cytometry or nanoparticle tracking analysis was used for MkEV counting. Both methods produced MkEVs that were relatively depleted of miR-486-5p and miR-22-3p cargo. However, while the shake flask-derived MkEVs were correspondingly less effective in promoting megakaryocytic differentiation of HSPCs, the syringe pump-derived MkEVs were more effective in doing so, suggesting the presence of unique, unidentified miRNA cargo components. Higher numbers of MkEVs were also produced by "older" Mk cultures, though miRNA cargo levels and MkEV bioactivity were unaffected by culture age. A reduction in MkEV production by Mks derived from late-differentiating HSPCs was also noted. Taken together, our results demonstrate that biomechanical force has an underappreciated and deeply influential role in MkEV biology, though that role may vary significantly depending on the nature of the force. Given the ubiquity of biomechanical force in vivo and in biomanufacturing, this phenomenon must be grappled with before MkEVs can attain clinical relevance.

Biomechanical control of lymphatic vessel physiology and functions.

The ever-growing research on lymphatic biology has clearly identified lymphatic vessels as key players that maintain human health through their functional roles in tissue fluid homeostasis, immunosurveillance, lipid metabolism and inflammation. It is therefore not surprising that the list of human diseases associated with lymphatic malfunctions has grown larger, including issues beyond lymphedema, a pathology traditionally associated with lymphatic drainage insufficiency. Thus, the discovery of factors and pathways that can promote optimal lymphatic functions may offer new therapeutic options. Accumulating evidence indicates that aside from biochemical factors, biomechanical signals also regulate lymphatic vessel expansion and functions postnatally. Here, we review how mechanical forces induced by fluid shear stress affect the behavior and functions of lymphatic vessels and the mechanisms lymphatic vessels employ to sense and transduce these mechanical cues into biological signals.

The role of biomechanical stress in extracellular vesicle formation, composition and activity.

Extracellular vesicles (EVs) are cornerstones of intercellular communication with exciting fundamental, clinical, and more broadly biotechnological applications. However, variability in EV composition, which results from the culture conditions used to generate the EVs, poses significant fundamental and applied challenges and a hurdle for scalable bioprocessing. Thus, an understanding of the relationship between EV production (and for clinical applications, manufacturing) and EV composition is increasingly recognized as important and necessary. While chemical stimulation and culture conditions such as cell density are known to influence EV biology, the impact of biomechanical forces on the generation, properties, and biological activity of EVs remains poorly understood. Given the omnipresence of these forces in EV preparation and in biomanufacturing, expanding the understanding of their impact on EV composition-and thus, activity-is vital. Although several publications have examined EV preparation and bioprocessing and briefly discussed biomechanical stresses as variables of interest, this review represents the first comprehensive evaluation of the impact of such stresses on EV production, composition and biological activity. We review how EV biogenesis, cargo, efficacy, and uptake are uniquely affected by various types, magnitudes, and durations of biomechanical forces, identifying trends that emerge both generically and for individual cell types. We also describe implications for scalable bioprocessing, evaluating processes inherent in common EV production and isolation methods, and propose a path forward for rigorous EV quality control.

Changes of Age-related Auricular Cartilage Plasticity and Biomechanical Property in a Rabbit Model.

OBJECTIVES: Ear molding is an emerging technique that can correct auricular deformities. Treatment initiation time is the most important prognostic determinant of ear molding. Here, we aimed to examine why auricular cartilage plasticity appeared to diminish with age. Thus, we characterized age-related changes in the biomechanical, biochemical, and morphological properties of auricular cartilage. METHODS: New Zealand rabbits were used as the experimental animal. We examined immature [postnatal 0 day (P0), 5 days (P5), 15 days (P15)], young [2 months (2M)], and mature [6 months (6M)] rabbits. Rabbits' ears were splinted and folded using adhesive fixation strips. Folding duration ranged from 1 day to 5 days to 10 days. Photographs were taken to calculate the retained fold angle. Cartilage morphology and extracellular matrix (ECM) content were examined histologically (using hematoxylin-eosin, Safranin O, elastic Van Gieson, and Masson's trichrome). Water content, DNA content, and cell density were also analyzed. Biomechanical properties were measured using a Nano indenter. RESULTS: Immature ears had smaller angles after strip removal, and the angled deformation lasted a longer time. Cartilage matrix compositions, including glycosaminoglycan (GAG), elastin fiber, and collagen, increased over development. The water content, DNA content, and cell density decreased with age. Young's modulus was significantly higher in mature cartilage. CONCLUSIONS: Here, we successfully established an animal model of ear molding and demonstrated that immature cartilage was associated with better plasticity. We also found that the cartilage's biomechanical property increased with the accumulation of ECM. The biomechanical change could underlie age-related shape plasticity. LEVEL OF EVIDENCE: NA Laryngoscope, 133:88-94, 2023.

Insights into intercellular receptor-ligand binding kinetics in cell communication.

Cell-cell communication is crucial for cells to sense, respond and adapt to environmental cues and stimuli. The intercellular communication process, which involves multiple length scales, is mediated by the specific binding of membrane-anchored receptors and ligands. Gaining insight into two-dimensional receptor-ligand binding kinetics is of great significance for understanding numerous physiological and pathological processes, and stimulating new strategies in drug design and discovery. To this end, extensive studies have been performed to illuminate the underlying mechanisms that control intercellular receptor-ligand binding kinetics via experiment, theoretical analysis and numerical simulation. It has been well established that the cellular microenvironment where the receptor-ligand interaction occurs plays a vital role. In this review, we focus on the advances regarding the regulatory effects of three factors including 1) protein-membrane interaction, 2) biomechanical force, and 3) bioelectric microenvironment to summarize the relevant experimental observations, underlying mechanisms, as well as their biomedical significances and applications. Meanwhile, we introduce modeling methods together with experiment technologies developed for dealing with issues at different scales. We also outline future directions to advance the field and highlight that building up systematic understandings for the coupling effects of these regulatory factors can greatly help pharmaceutical development.

RhoA-ROCK competes with YAP to regulate amoeboid breast cancer cell migration in response to lymphatic-like flow.

Lymphatic drainage generates force that induces prostate cancer cell motility via activation of Yes-associated protein (YAP), but whether this response to fluid force is conserved across cancer types is unclear. Here, we show that shear stress corresponding to fluid flow in the initial lymphatics modifies taxis in breast cancer, whereas some cell lines use rapid amoeboid migration behavior in response to fluid flow, a separate subset decrease movement. Positive responders displayed transcriptional profiles characteristic of an amoeboid cell state, which is typical of cells advancing at the edges of neoplastic tumors. Regulation of the HIPPO tumor suppressor pathway and YAP activity also differed between breast subsets and prostate cancer. Although subcellular localization of YAP to the nucleus positively correlated with overall velocity of locomotion, YAP gain- and loss-of-function demonstrates that YAP inhibits breast cancer motility but is outcompeted by other pro-taxis mediators in the context of flow. Specifically, we show that RhoA dictates response to flow. GTPase activity of RhoA, but not Rac1 or Cdc42 Rho family GTPases, is elevated in cells that positively respond to flow and is unchanged in cells that decelerate under flow. Disruption of RhoA or the RhoA effector, Rho-associated kinase (ROCK), blocked shear stress-induced motility. Collectively, these findings identify biomechanical force as a regulator amoeboid cell migration and demonstrate stratification of breast cancer subsets by flow-sensing mechanotransduction pathways.

Epidemiologic, Postmortem Computed Tomography-Morphologic and Biomechanical Analysis of the Effects of Non-Invasive External Pelvic Stabilizers in Genuine Unstable Pelvic Injuries.

Unstable pelvic injuries are rare (3-8% of all fractures) but are associated with a mortality of up to 30%. An effective way to treat venous and cancellous sources of bleeding prehospital is to reduce intrapelvic volume with external noninvasive pelvic stabilizers. Scientifically reliable data regarding pelvic volume reduction and applicable pressure are lacking. Epidemiologic data were collected, and multiple post-mortem CT scans and biomechanical measurements were performed on real, unstable pelvic injuries. Unstable pelvic injury was shown to be the leading source of bleeding in only 19%. All external non-invasive pelvic stabilizers achieved intrapelvic volume reduction; the T-POD® succeeded best on average (333 ± 234 cm3), but with higher average peak traction (110 N). The reduction results of the VBM® pneumatic pelvic sling consistently showed significantly better results at a pressure of 200 mmHg than at 100 mmHg at similar peak traction forces. All pelvic stabilizers exhibited the highest peak tensile force shortly after application. Unstable pelvic injuries must be considered as an indicator of serious concomitant injuries. Stabilization should be performed prehospital with specific pelvic stabilizers, such as the T-POD® or the VBM® pneumatic pelvic sling. We recommend adjusting the pressure recommendation of the VBM® pneumatic pelvic sling to 200 mmHg.

Visualizing the Invisible: Advanced Optical Microscopy as a Tool to Measure Biomechanical Forces.

The importance of mechanical force in biology is evident across diverse length scales, ranging from tissue morphogenesis during embryo development to mechanotransduction across single adhesion proteins at the cell surface. Consequently, many force measurement techniques rely on optical microscopy to measure forces being applied by cells on their environment, to visualize specimen deformations due to external forces, or even to directly apply a physical perturbation to the sample via photoablation or optogenetic tools. Recent developments in advanced microscopy offer improved approaches to enhance spatiotemporal resolution, imaging depth, and sample viability. These advances can be coupled with already existing force measurement methods to improve sensitivity, duration and speed, amongst other parameters. However, gaining access to advanced microscopy instrumentation and the expertise necessary to extract meaningful insights from these techniques is an unavoidable hurdle. In this Live Cell Imaging special issue Review, we survey common microscopy-based force measurement techniques and examine how they can be bolstered by emerging microscopy methods. We further explore challenges related to the accompanying data analysis in biomechanical studies and discuss the various resources available to tackle the global issue of technology dissemination, an important avenue for biologists to gain access to pre-commercial instruments that can be leveraged for biomechanical studies.

Biomechanical Regulation of Hematopoietic Stem Cells in the Developing Embryo.

PURPOSE OF REVIEW: The contribution of biomechanical forces to hematopoietic stem cell (HSC) development in the embryo is a relatively nascent area of research. Herein, we address the biomechanics of the endothelial-to-hematopoietic transition (EHT), impact of force on organelles, and signaling triggered by extrinsic forces within the aorta-gonad-mesonephros (AGM), the primary site of HSC emergence. RECENT FINDINGS: Hemogenic endothelial cells undergo carefully orchestrated morphological adaptations during EHT. Moreover, expansion of the stem cell pool during embryogenesis requires HSC extravasation into the circulatory system and transit to the fetal liver, which is regulated by forces generated by blood flow. Findings from other cell types also suggest that forces external to the cell are sensed by the nucleus and mitochondria. Interactions between these organelles and the actin cytoskeleton dictate processes such as cell polarization, extrusion, division, survival, and differentiation. SUMMARY: Despite challenges of measuring and modeling biophysical cues in the embryonic HSC niche, the past decade has revealed critical roles for mechanotransduction in governing HSC fate decisions. Lessons learned from the study of the embryonic hematopoietic niche promise to provide critical insights that could be leveraged for improvement in HSC generation and expansion ex vivo.

Mechanoregulation in hematopoiesis and hematologic disorders.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) are reliant on intrinsic and extrinsic factors for tight control of self-renewal, quiescence, differentiation, and homing. Given the intimate relationship between HSCs and their niche, increasing numbers of studies are examining how biophysical cues in the hematopoietic microenvironment impact HSC functions. RECENT FINDINGS: Numerous mechanosensors are present on hematopoietic cells, including integrins, mechanosensitive ion channels, and primary cilia. Integrin-ligand adhesion, in particular, has been found to be critical for homing and anchoring of HSCs and progenitors in the bone marrow. Integrin-mediated interactions with ligands present on extracellular matrix and endothelial cells are key to establishing long-term engraftment and quiescence of HSCs. Importantly, disruption in the architecture and cellular composition of the bone marrow associated with conditioning regimens and primary myelofibrosis exposes HSCs to a profoundly distinct mechanical environment, with potential implications for progression of hematologic dysfunction and pathologies. SUMMARY: Study of the mechanobiological signals that govern hematopoiesis represents an important future step toward understanding HSC biology in homeostasis, aging, and cancer.

The long noncoding RNA H19 attenuates force-driven cartilage degeneration via miR-483-5p/Dusp5.

Developmental dysplasia of the hip (DDH) is a common hip disease characterized by abnormal development of the acetabulum and femoral head. In most cases, DDH ultimately leads to osteoarthritis. Anomalous biomechanical force plays an important role in cartilage degeneration in DDH. However, in addition to mechanical wear, the underlying molecular mechanisms in cartilage degeneration in DDH remain unclear. This study analyzed the effect of long noncoding RNA (lncRNA)-H19 on DDH cartilage degradation. To elucidate the specific role of lncRNA H19, we established an intermittent cyclic mechanical stress (ICMS) cell force model to simulate abnormal biomechanical environment in vitro. Then, the roles of lncRNA-H19 were also determined in vivo by establishing a model of swaddling DDH. We observed that patients with DDH possessed low levels of lncRNA-H19, COL2A1, and Aggrecan but high levels of MMP3 and Adamts5. The same results were also obtained in a DDH rat model. Furthermore, the data suggested that ICMS promoted cartilage degeneration and caused reorientation of the cytoskeleton, and lncRNA H19 helped inhibit cartilage degeneration. Bioinformatics analysis and lncRNA sequencing were performed, and luciferase assays showed that lncRNA H19 and Dusp5 are both direct targets of miR-483-5p. Moreover, Dups5 plays a negative role in ICMS-induced cartilage degradation by activating the Erk and p38 pathways. In vivo, lncRNA H19 had protective effects on the swaddling DDH model. These findings indicate that lncRNA-H19 played a positive role in cartilage degradation in DDH through the lncRNA H19/miR-483-5p/Dusp5 axis.

TAZ responds to fluid shear stress to regulate the cell cycle.

Physical forces associated with tumor growth and drainage alter cancer cell invasiveness and metastatic potential. We previously showed that fluid frictional force, or shear stress, typical of lymphatic flow induces YAP1/TAZ activation in prostate cancer cells to promote motility dependent upon YAP1 but not TAZ. Here, we show that shear stress elevates TAZ protein levels and promotes TAZ nuclear localization. Increased TAZ activity drives increased DNA synthesis and induces AMOTL2, ANKRD1, and CTGF gene transcription independently of YAP1. Ectopic expression of constitutively activated TAZ increases expression of these TAZ target genes and promotes cell proliferation of prostate cancer cells. Conversely, silencing of TAZ results in reduced proliferation. Together, our data show that force-induced TAZ regulates signaling that dictates cell division, and suggest that TAZ may govern cellular proliferation of cancer cells traveling through the lymphatics in response to biophysical cues.

A Co-culture Assay to Determine Efficacy of TNF-α Suppression by Biomechanically Induced Human Bone Marrow Mesenchymal Stem Cells.

The beneficial effects of mesenchymal stem cell (MSC)-based cellular therapies are believed to be mediated primarily by the ability odansf MSCs to suppress inflammation associated with chronic or acute injury, infection, autoimmunity, and graft-versus-host disease. To specifically address the effects of frictional force caused by blood flow, or wall shear stress (WSS), on human MSC immunomodulatory function, we have utilized microfluidics to model WSS at the luminal wall of arteries. Anti-inflammatory potency of MSCs was subsequently quantified via measurement of TNF-α production by activated murine splenocytes in co-culture assays. The TNF-α suppression assay serves as a reproducible platform for functional assessment of MSC potency and demonstrates predictive value as a surrogate assay for MSC therapeutic efficacy.

Biomechanical Forces Promote Immune Regulatory Function of Bone Marrow Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are believed to mobilize from the bone marrow in response to inflammation and injury, yet the effects of egress into the vasculature on MSC function are largely unknown. Here we show that wall shear stress (WSS) typical of fluid frictional forces present on the vascular lumen stimulates antioxidant and anti-inflammatory mediators, as well as chemokines capable of immune cell recruitment. WSS specifically promotes signaling through NFκB-COX2-prostaglandin E2 (PGE2 ) to suppress tumor necrosis factor-α (TNF-α) production by activated immune cells. Ex vivo conditioning of MSCs by WSS improved therapeutic efficacy in a rat model of traumatic brain injury, as evidenced by decreased apoptotic and M1-type activated microglia in the hippocampus. These results demonstrate that force provides critical cues to MSCs residing at the vascular interface which influence immunomodulatory and paracrine activity, and suggest the potential therapeutic use of force for MSC functional enhancement. Stem Cells 2017;35:1259-1272.

Biomechanical force induces the growth factor production in human periodontal ligament-derived cells.

Although many reports have been published on the functional roles of periodontal ligament (PDL) cells, the mechanisms involved in the maintenance and homeostasis of PDL have not been determined. We investigated the effects of biomechanical force on growth factor production, phosphorylation of MAPKs, and intracellular transduction pathways for growth factor production in human periodontal ligament (hPDL) cells using MAPK inhibitors. hPDL cells were exposed to mechanical force (6 MPa) using a hydrostatic pressure apparatus. The levels of growth factor mRNA and protein were examined by real-time RT-PCR and ELISA. The phosphorylation of MAPKs was measured using BD™ CBA Flex Set. In addition, MAPKs inhibitors were used to identify specific signal transduction pathways. Application of biomechanical force (equivalent to occlusal force) increased the synthesis of VEGF-A, FGF-2, and NGF. The application of biomechanical force increased the expression levels of phosphorylated ERK and p38, but not of JNK. Furthermore, the levels of VEGF-A and NGF expression were suppressed by ERK or p38 inhibitor. The growth factors induced by biomechanical force may play a role in the mechanisms of homeostasis of PDL.

Biomechanical force in blood development: extrinsic physical cues drive pro-hematopoietic signaling.

The hematopoietic system is dynamic during development and in adulthood, undergoing countless spatial and temporal transitions during the course of one's life. Microenvironmental cues in the many unique hematopoietic niches differ, characterized by distinct soluble molecules, membrane-bound factors, and biophysical features that meet the changing needs of the blood system. Research from the last decade has revealed the importance of substrate elasticity and biomechanical force in determination of stem cell fate. Our understanding of the role of these factors in hematopoiesis is still relatively poor; however, the developmental origin of blood cells from the endothelium provides a model for comparison. Many endothelial mechanical sensors and second messenger systems may also determine hematopoietic stem cell fate, self renewal, and homing behaviors. Further, the intimate contact of hematopoietic cells with mechanosensitive cell types, including osteoblasts, endothelial cells, mesenchymal stem cells, and pericytes, places them in close proximity to paracrine signaling downstream of mechanical signals. The objective of this review is to present an overview of the sensors and intracellular signaling pathways activated by mechanical cues and highlight the role of mechanotransductive pathways in hematopoiesis.