Calpain- and talin-dependent control of microvascular pericyte contractility and cellular stiffness

Pericytes surround capillary endothelial cells and exert contractile forces modulating microvascular tone and endothelial growth. We previously described pericyte contractile phenotype to be Rho GTPase- and α-smooth muscle actin (αSMA)-dependent. However, mechanisms mediating adhesion-dependent shape changes and contractile force transduction remain largely equivocal. We now report that the neutral cysteine protease, calpain, modulates pericyte contractility and cellular stiffness via talin, an integrin-binding and F-actin associating protein. Digital imaging and quantitative analyses of living cells reveal significant perturbations in contractile force transduction detected via deformation of silicone substrata, as well as perturbations of mechanical stiffness in cellular contractile subdomains quantified via atomic force microscope (AFM)-enabled nanoindentation. Pericytes overexpressing GFP-tagged talin show significantly enhanced contractility (~ two-fold), which is mitigated when either the calpain-cleavage resistant mutant talin L432G or vinculin are expressed. Moreover, the cell-penetrating, calpain-specific inhibitor termed CALPASTAT reverses talin-enhanced, but not Rho GTP-dependent, contractility. Interestingly, our analysis revealed that CALPASTAT, but not its inactive mutant, alters contractile cell-driven substrata deformations while increasing mechanical stiffness of subcellular contractile regions of these pericytes. Altogether, our results reveal that calpain-dependent cleavage of talin modulates cell contractile dynamics, which in pericytes may prove instrumental in controlling normal capillary function or microvascular pathophysiology.National Science Foundation (U.S.) (CAREER Award)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowshi

Macromolecular Crowding Directs Extracellular Matrix Organization and Mesenchymal Stem Cell Behavior

Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro

International Consensus Statement on Rhinology and Allergy: Rhinosinusitis

Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR‐RS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICAR‐RS‐2021 as well as updates to the original 140 topics. This executive summary consolidates the evidence‐based findings of the document. Methods: ICAR‐RS presents over 180 topics in the forms of evidence‐based reviews with recommendations (EBRRs), evidence‐based reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICAR‐RS‐2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidence‐based management algorithm is provided. Conclusion: This ICAR‐RS‐2021 executive summary provides a compilation of the evidence‐based recommendations for medical and surgical treatment of the most common forms of RS

Macromolecular crowding directly alters organization of deposited extracellular matrix proteins and thus alters the orientation of the actin cytoskeleton.

<p>(<b>A</b>) Immunostaining of intracellular F-actin (red), intracellular vinculin (green) as a focal adhesion protein involved in the linking of integrin to actin cytoskeleton, and nucleus (blue, DAPI) of human bone marrow-derived mesenchymal stromal or stem cells (MSCs) after 3 days of cell culture in media containing macromolecular crowders (+MMC media) and (<b>B</b>) −MMC media. Scale bars = 30 µm. (<b>C</b>) Quantification of average angular standard deviation for F-actin (N = 10 +MMC, N = 10 −MMC, p = 0.0223) where lower values indicate a higher degree of alignment. (<b>D</b>) Effective Young's elastic modulus in kPa measured by atomic force microscopy enabled nanoindentation of MSCs ± MMC suggests a stiffening of the cortical cytoskeleton +MMC. (<b>E</b>) Average angular standard deviation of FITC-conjugated rat tail type-I collagen network deposited on plasma treated glass coverslips, (<b>F</b>) in media absent of macromolecular crowders (−MMC) and, (<b>G</b>) +MMC. Scale bars = 25 µm. (<b>H</b>) Immunostaining of F-actin (red) after 3 days for human bone marrow-derived mesenchymal stromal or stem cells cultured in basal −MMC media seeded onto type-I collagen networks formed under −MMC, or (<b>I</b>) +MMC conditions (N = 13 +MMC, N = 13 −MMC, p = 0.0001). Scale bars = 25 µm. (<b>J</b>) Average angular standard deviation of actin fibers for <b>H</b> and <b>I</b>. Values are reported as mean ± standard error of measurement. * indicates statistical significance (p<0.05). ** indicates statistical significance (p<0.001).</p

Consequences of altered extracellular matrix organization include enhanced proliferation, altered adhesion, and decreased migration.

<p>(<b>A</b>) Human bone marrow-derived mesenchymal stromal or stem cells (MSCs) cultured in media containing macromolecular crowders (+MMC media, filled red circle) demonstrate statistically significant increase in proliferation versus cells in −MMC media (filled blue square). Furthermore, cells seeded directly in +MMC media (filled green triangle) also demonstrate a similar increase in proliferation. (<b>B</b>) Cells seeded directly in +MMC media (green) demonstrate enhanced adhesion after 24 hours as compared to cells seeded without crowder (−MMC, blue +MMC (24 h post seeding), red). (<b>C</b>) MSCs cultured in basal media on collagen type-I deposited +MMC (open red circle) also exhibit similarly altered proliferation behavior compared to MSCs on collagen deposited −MMC (open blue square). (<b>D</b>) Migration of cells, as determined by a wound healing assay, is significantly reduced +MMC (open red circle) (<b>E</b>) as compared to −MMC (open blue square) (<b>F</b>) (N = 9). Values are reported as mean ± standard error of measurement. * indicates statistical significance (p<0.001).</p

Macromolecular crowding induces alignment of extracellular matrix fibers and an increase in deposition of collagen type-I in human bone marrow-derived mesenchymal stromal or stem cells (MSCs).

<p>(<b>A</b>) Atomic force microscopy (AFM) contact mode deflection images of extracellular matrix deposited by MSCs left behind after detergent removal of cells in media containing macromolecular crowders (+MMC media) and (<b>B</b>) −MMC media after 7 days. Scale bars = 2 mm. (<b>C</b>) Average angular standard deviation for AFM imaging of extracellular matrix in <b>A</b> and <b>B</b> (N = 5 +MMC, N = 5 −MMC, p = 0.1363). (<b>D</b>) Immunostaining of extracellular fibronectin (red) and cell nucleus (blue, DAPI) for +MMC and (<b>E</b>) −MMC in MSCs after 3 days of culture. Scale bars = 30 µm. (<b>F</b>) Average angular standard deviation for fibronectin in <b>D</b> and <b>E</b> (N = 15 +MMC, N = 11 −MMC, p = 0.0013). (<b>G</b>) Immunostaining of extracellular collagen IV (green) and cell nucleus (blue, DAPI) +MMC, (<b>H</b>) and −MMC, in hMSCs after 3 days of culture. Scale bars = 15 mm (<b>I</b>) Average angular standard deviation for collagen IV in <b>G</b> and <b>H</b> (N = 12 +MMC, N = 8 −MMC, p = 0.0253). Lower values of average angular standard deviation indicate a higher degree of alignment of fibers comprising the extracellular matrix. (<b>J</b>) Western blot of collagen type-I secreted by human bone marrow-derived mesenchymal stromal or stem cells after 48 hrs in culture medium ± MMC demonstrates a significant increase in the cell deposited collagen into the matrix +MMC. (<b>K</b>) Normalized densitometry of collagen type-I Western blot demonstrates a shift in collagen distribution from media to matrix, indicative of the enhanced deposition of collagen in the presence of MMCs. Significant increases in crosslinked collagen α-chains (β-bands) is observed in the cell deposited matrix +MMC, as compared to −MMC conditions (p<0.0001). Values are reported as mean ± standard error of measurement. * indicates p = 0.1363; and ** indicates statistical significance (p<0.05).</p

Schematic of cell and matrix reciprocity under macromolecular crowding.

<p>(<b>A</b>) Adherent mesenchymal stromal cell (MSC) under typical <i>in vitro</i> culture conditions. (<b>B</b>) Upon the addition of macromolecular crowders, excluded volume effects promote bundling and alignment of extracellular matrix (ECM) fibers. (<b>C</b>) In response to the aligned ECM, the intracellular actin cytoskeleton reorganizes to align with the ECM. In addition, the cell secretes more matrix proteins, which are deposited to and further enhance the alignment of the ECM. We find that these reciprocal changes in the matrix material and intracellular cytoskeletal organization correlate with an increase in proliferation and a decrease in motility of MSCs.</p

Autocrine-controlled formation and function of tissue-like aggregates by hepatocytes in micropatterned hydrogel arrays

The liver carries out a variety of essential functions regulated in part by autocrine signaling, including hepatocyte-produced growth factors and extracellular matrix (ECM). The local concentrations of autocrine factors are governed by a balance between receptor-mediated binding at the cell surface and diffusion into the local matrix and are thus expected to be influenced by the dimensionality of the cell culture environment. To investigate the role of growth factor and ECM-modulated autocrine signaling in maintaining appropriate primary hepatocyte survival, metabolic functions, and polarity, we created three-dimensional cultures of defined geometry using micropatterned semisynthetic polyethylene glycol–fibrinogen hydrogels to provide a mechanically compliant, nonadhesive material platform that could be modified by cell-secreted factors. We found that in the absence of exogenous peptide growth factors or ECM, hepatocytes retain the epidermal growth factor (EGF) receptor ligands (EGF and transforming growth factor-α) and the proto-oncogenic mesenchymal epithelial transition factor (c-MET) ligand hepatocyte growth factor (HGF), along with fibronectin. Further, hepatocytes cultured in this three-dimensional microenvironment maintained high levels of liver-specific functions over the 10-day culture period. Function-blocking inhibitors of α5β1 or EGF receptor dramatically reduced cell viability and function, suggesting that signaling by both these receptors is needed for in vitro survival and function of hepatocytes in the absence of other exogenous signals.National Institutes of Health (U.S.) (grant NIBIB R01EB003805)National Institute of Environmental Health Sciences (MIT Center for Environmental Health Sciences (P30ES002109))National Institute of Environmental Health Sciences (MIT Center for Environmental Health Sciences (MIT Center for Environmental Health Sciences (R01ES015241)Ruth L. Kirschstein National Research Service Award (postdoctoral fellowship)National Science Foundation (U.S.) (CAREER Award)National Defense Science and Engineering Graduate Fellowshi