Charles K. Thodeti

Tumor-derived endothelial cells exhibit aberrant Rho-mediated mechanosensing and abnormal angiogenesis in vitro

Tumor blood vessels exhibit abnormal structure and function that cause disturbed blood flow and high interstitial pressure, which impair delivery of anti-cancer agents. Past efforts to normalize the tumor vasculature have focused on inhibition of soluble angiogenic factors, such as VEGF; however, capillary endothelial (CE) cell growth and differentiation during angiogenesis are also influenced by mechanical forces conveyed by the extracellular matrix (ECM). Here, we explored the possibility that tumor CE cells form abnormal vessels because they lose their ability to sense and respond to these physical cues. These studies reveal that, in contrast to normal CE cells, tumor-derived CE cells fail to reorient their actin cytoskeleton when exposed to uniaxial cyclic strain, exhibit distinct shape sensitivity to variations in ECM elasticity, exert greater traction force, and display an enhanced ability to retract flexible ECM substrates and reorganize into tubular networks in vitro. These behaviors correlate with a constitutively high level of baseline activity of the small GTPase Rho and its downstream effector, Rho-associated kinase (ROCK). Moreover, decreasing Rho-mediated tension by using the ROCK inhibitor, Y27632, can reprogram the tumor CE cells so that they normalize their reorientation response to uniaxial cyclic strain and their ability to form tubular networks on ECM gels. Abnormal Rho-mediated sensing of mechanical cues in the tumor microenvironment may therefore contribute to the aberrant behaviors of tumor CE cells that result in the development of structural abnormalities in the cancer microvasculature.

Directional control of cell motility through focal adhesion positioning and spatial control of Rac activation

Local physical interactions between cells and extracellular matrix (ECM) influence directional cell motility that is critical for tissue development, wound repair, and cancer metastasis. Here we test the possibility that the precise spatial positioning of focal adhesions governs the direction in which cells spread and move. NIH 3T3 cells were cultured on circular or linear ECM islands, which were created using a microcontact printing technique and were 1 μm wide and of various lengths (1 to 8 μm) and separated by 1 to 4.5 μm wide nonadhesive barrier regions. Cells could be driven proactively to spread and move in particular directions by altering either the interisland spacing or the shape of similar‐sized ECM islands. Immunofluorescence microscopy confirmed that focal adhesions assembled preferentially above the ECM islands, with the greatest staining intensity being observed at adhesion sites along the cell periphery. Rac‐FRET analysis of living cells revealed that Rac became activated within 2 min after peripheral membrane extensions adhered to new ECM islands, and this activation wave propagated outward in an oriented manner as the cells spread from island to island. A computational model, which incorporates that cells preferentially protrude membrane processes from regions near newly formed focal adhesion contacts, could predict with high accuracy the effects of six different arrangements of micropatterned ECM islands on directional cell spreading. Taken together, these results suggest that physical properties of the ECM may influence directional cell movement by dictating where cells will form new focal adhesions and activate Rac and, hence, govern where new membrane protrusions will form.— Xia N., Thodeti, C. K., Hunt, T. P., Xu, Q., Ho, M., Whitesides, G. M., Westervelt, R., Ingber D. E. Directional control of cell motility through focal adhesion positioning and spatial control of Rac activation. FASEB J. 22, 1649–1659 (2008)

Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface β1 integrins

Integrins are ubiquitous transmembrane mechanoreceptors that elicit changes in intracellular biochemistry in response to mechanical force application, but these alterations generally proceed over seconds to minutes. Stress-sensitive ion channels represent another class of mechanoreceptors that are activated much more rapidly (within msec), and recent findings suggest that calcium influx through Transient Receptor Potential Vanilloid-4 (TRPV4) channels expressed in the plasma membrane of bovine capillary endothelial cells is required for mechanical strain-induced changes in focal adhesion assembly, cell orientation and directional migration. However, whether mechanically stretching a cells extracellular matrix (ECM) adhesions might directly activate cell surface ion channels remains unknown. Here we show that forces applied to beta1 integrins result in ultra-rapid (within 4 msec) activation of calcium influx through TRPV4 channels. The TRPV4 channels were specifically activated by mechanical strain in the cytoskeletal backbone of the focal adhesion, and not by deformation of the lipid bilayer or submembranous cortical cytoskeleton alone. This early-immediate calcium signaling response required the distal region of the beta1 integrin cytoplasmic tail that contains a binding site for the integrin-associated transmembrane CD98 protein, and external force application to CD98 within focal adhesions activated the same ultra-rapid calcium signaling response. Local direct strain-dependent activation of TRPV4 channels mediated by force transfer from integrins and CD98 may therefore enable compartmentalization of calcium signaling within focal adhesions that is critical for mechanical control of many cell behaviors that underlie cell and tissue development.

Investigating complexity of protein-protein interactions in focal adhesions

The formation of focal adhesions governs cell shape and function; however, there are few measurements of the binding kinetics of focal adhesion proteins in living cells. Here, we used the fluorescence recovery after photobleaching (FRAP) technique, combined with mathematical modeling and scaling analysis to quantify dissociation kinetics of focal adhesion proteins in capillary endothelial cells. Novel experimental protocols based on mathematical analysis were developed to discern the rate-limiting step during FRAP. Values for the dissociation rate constant k(OFF) ranged over an order of magnitude from 0.009+/-0.001/s for talin to 0.102+/-0.010/s for FAK, indicating that talin is bound more strongly than other proteins in focal adhesions. Comparisons with in vitro measurements reveal that multiple focal adhesion proteins form a network of bonds, rather than binding in a pair-wise manner in these anchoring structures in living cells.

Force meets chemistry: Analysis of mechanochemical conversion in focal adhesions using fluorescence recovery after photobleaching

Mechanotransduction—the process by which mechanical forces are converted into changes of intracellular biochemistry—is critical for normal cell and tissue function. Integrins facilitate mechanochemical conversion by transferring physical forces from the extracellular matrix, across the cell surface, and to cytoskeletal‐associated proteins within focal adhesions. It is likely that force alters biochemistry at these sites by altering molecular binding affinities of a subset of focal adhesion proteins, but this has been difficult to quantify within living cells. Here, we describe how the fluorescence recovery after photobleaching (FRAP) technique can be adapted and used in conjunction with mathematical models to directly measure force‐dependent alterations in molecular binding and unbinding rate constants of individual focal adhesion proteins in situ. We review these recent findings, and discuss the strengths and limitations of this approach for analysis of mechanochemical signaling in focal adhesions and other cellular structures. The ability to quantify molecular binding rate constants in the physical context of the living cytoplasm should provide new insight into the molecular basis of cellular mechanotransduction. It also may facilitate future efforts to bridge biological experimentation and mathematical modeling in our quest for a systems biology level description of cell regulation. J. Cell. Biochem. 97: 1175–1183, 2006.

Mechanical control of cAMP signaling through integrins is mediated by the heterotrimeric Gαs protein

Mechanical stresses that are preferentially transmitted across the cell surface via transmembrane integrin receptors activate gene transcription by triggering production of intracellular chemical second messengers, such as cAMP. Here we show that the sensitivity of the cAMP signaling pathway to mechanical stresses transferred across β1 integrins is mediated by force‐dependent activation of the heterotrimeric G protein subunit Gαs within focal adhesions at the site of stress application. Gαs is recruited to focal adhesions that form within minutes following clustering of β1 integrins induced by cell binding to magnetic microbeads coated with activating integrin ligands, and β1 integrin and Gαs co‐precipitate when analyzed biochemically. Stress application to activated β1 integrins using magnetic twisting cytometry increases Gαs recruitment and activates these large G proteins within focal adhesions, as measured by binding of biotinylated azido‐anilido‐GTP, whereas application of similar stresses to inactivated integrins or control histocompatibility antigens has little effect. This response is relevant physiologically as application of mechanical strain to cells bound to flexible extracellular matrix‐coated substrates induce translocation of phospho‐CREB to the nucleus, which can be attenuated by inhibiting Gαs activity, either using the inhibitor melittin or suppressing its expression using siRNA. Although integrins are not typical G protein‐coupled receptors, these results show that integrins focus mechanical stresses locally on heterotrimeric G proteins within focal adhesions at the site of force application, and transduce mechanical stimuli into an intracellular cAMP signaling response by activating Gαs at these membrane signaling sites. J. Cell. Biochem. 106: 529–538, 2009.

Paxillin mediates sensing of physical cues and regulates directional cell motility by controlling lamellipodia positioning.

Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5–10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax−/− and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax−/− cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax−/− and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices.

Activation of mechanosensitive ion channel TRPV4 normalizes tumor vasculature and improves cancer therapy

Tumor vessels are characterized by abnormal morphology and hyperpermeability that together cause inefficient delivery of chemotherapeutic agents. Although vascular endothelial growth factor has been established as a critical regulator of tumor angiogenesis, the role of mechanical signaling in the regulation of tumor vasculature or tumor endothelial cell (TEC) function is not known. Here we show that the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) regulates tumor angiogenesis and tumor vessel maturation via modulation of TEC mechanosensitivity. We found that TECs exhibit reduced TRPV4 expression and function, which is correlated with aberrant mechanosensitivity towards extracellular matrix stiffness, increased migration and abnormal angiogenesis by TEC. Further, syngeneic tumor experiments revealed that the absence of TRPV4 induced increased vascular density, vessel diameter and reduced pericyte coverage resulting in enhanced tumor growth in TRPV4 knockout mice. Importantly, overexpression or pharmacological activation of TRPV4 restored aberrant TEC mechanosensitivity, migration and normalized abnormal angiogenesis in vitro by modulating Rho activity. Finally, a small molecule activator of TRPV4, GSK1016790A, in combination with anticancer drug cisplatin, significantly reduced tumor growth in wild-type mice by inducing vessel maturation. Our findings demonstrate TRPV4 channels to be critical regulators of tumor angiogenesis and represent a novel target for anti-angiogenic and vascular normalization therapies.

Micromechanical Design Criteria for Tissue Engineering Biomaterials

A key goal in the field of tissue engineering is the development of novel biomimetic materials that mimic the features of natural materials responsible for normal control of multicellular assembly, and seamlessly integrate with living tissues. Past work in this area has focused on the optimization of biomaterial chemistry and selection of appropriate biological cues (e.g., morphogens, ECM-derived adhesive ligands) to promote effective tissue development. However, tissue formation is also influenced by microscale mechanical forces that cells generate in their contractile cytoskeleton and exert on their adhesions to extracellular matrix (ECM) and to neighboring cells. Cells also sense these forces through transmembrane adhesion receptors, such as integrins and cadherins, which focus mechanical stresses on load-bearing molecular structures within membrane adhesion complexes (e.g., focal adhesions and junctional complexes) and linked cytoskeletal filaments and nuclear scaffolds. Resulting stress-induced changes in molecular shape and function mediate mechanotransduction – the conversion of a mechanical signal into an intracellular biochemical response – ultimately leading to integrated changes in global cell behavior that drive tissue patterning. Here, we highlight the key role that mechanical forces play in tissue development, and discuss how this knowledge might be leveraged to create a new set of physical design criteria for the development of novel biomimetic materials for regenerative medicine.

Mechanisms of Tumor Cell Migration and Invasion in Lung Cancer Metastasis

Cancer metastasis is a multistep process that involves tumor cell migration and invasion through tumor stroma, intravasation into and extravasation out of the blood vessels, and accumulation at a distant organ site. These events arise from concomitant alterations in the genetic, chemical, and physical state of tumor cells and its microenvironment. This chapter will, however, focus on the molecular determinants of tumor cell migration and invasion, with special emphasis on the cross talk between extracellular matrix, integrin receptors, matrix metalloproteases, and Rho GTPases, all of which undergo dynamic regulation to ultimately modulate cell shape and tension and, thereby, their migratory and invasive behavior. Elucidating these molecular mechanisms will likely identify key players in the metastatic process, which can be exploited to develop novel therapeutic strategies for the treatment for lung cancer.