This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation. The interstitial fluid pressure (PIF) is of importance for control of tissue fluid balance. A lowering of PIF in vivo will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling PIF. Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of PIF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of PIF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored PIF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered PIF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered PIF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from Streptococcus equi modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction in vitro and control of PIF in vivo, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence PIF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.

All blood vessels in the microvasculature are embedded in loose connective tissue, which regulates the transport of fluid to and from tissues. The intersti-tial fluid pressure (IFP) is one of the forces that control this transport. A lowering of IFP in vivo results in an increased transport of fluid from the circulation into the underhydrated connective tissues, resulting in edema formation. During homeostasis, contractile connective tissue cells exert a tension on the connective tissue fibrous network by binding with β1 in-tegrins, thereby actively controlling IFP. During inflammation, the IFP is lowered but platelet-derived growth factor (PDGF)-BB induces an IFP nor-malization dependent on integrin αVβ3. We demonstrate that extracellular proteins from Streptococcus equi subspecies equi modulated cell-mediated and integrin αVβ3-directed collagen gel contraction in vitro. One of these proteins, the collagen- and fibronectin binding FNE, stimulated contraction by a process dependent on fibronectin synthesis. This study identified a pos-sible novel virulence mechanism for bacteria based on the ability of bacteria to modulate the edema response. Another protein, the collagen-binding pro-tein CNE, inhibited contraction and this led to the identification of sites in collagen monomers that potentially are involved in connecting αVβ3 to the collagen network. PDGF-BB and prostaglandin E1 (PGE1) stimulate and inhibit collagen gel contraction in vitro and normalize and lower IFP, respec-tively. We showed that these agents affected both similar and different sets of actin-binding proteins. PDGF-BB stimulated actin cytoskeleton dynamics whereas PGE1 inhibited processes dependent on cytoskeletal motor and adhesive functions, suggesting that these different activities may partly ex-plain the contrasting effects of PGE1 and PDGF-BB on contraction and IFP. Mutation of the phosphatidylinositol 3’-kinase (PI3K), but not phospholipase C (PLC)γ activation site, rendered cells unable to respond to PDGF-BB in contraction and in activation of the actin binding and severing protein cofilin. Ability to activate cofilin after PDGF-BB stimulation correlated with ability to respond to PDGF-BB in contraction, suggesting a role for cofilin in this process downstream of PDGF receptor-activated PI3K. Many proteins can modulate contraction either by affecting the extracellular matrix and cell adhesions or by altering cytoskeletal dynamics. Knowledge on how these proteins might influence IFP is likely to be of clinical importance for treat-ment of inflammatory conditions including anaphylaxis, septic shock and also carcinoma growth.

Background: Patients with burns are susceptible to organ failure, and there is indirect evidence that leukocytes may contribute to this process. They may change the expression of cell-surface receptors after certain stimuli, for example, the burn. We therefore aimed to assess the changes induced by the burn in the expression of leukocyte cell-surface receptors CD11b, CD14, CD16, and CD62L on the surface of PMNs and monocytes. We also wanted to examine the dynamics of this activation during the first week after the burn, and to relate it to the size of the injury. Methods: Ten patients with burns of andgt;15% (TBSA) were included in the study. Blood samples were collected on arrival and every consecutive morning during the first week. Healthy volunteers acted as controls. Results: PMN CD11b expression was increased. The extent of PMN CD11b expression correlated negatively to the size of the full thickness burn. Monocyte CD14 expression increased initially but there was no relation to the size of the burn. PMN CD16 expression decreased initially during the first days and the decrease was related to burn size. CD62L did not vary depending on the burn in either PMN or monocytes during the first week after the burn. Conclusion: This study showed that specific receptors on the surface of leukocytes (PMN CD11b, monocyte CD14 and PMN CD16) are affected by the burn. Expression of PMN CD11b and CD16 are related to burn size. Burn-induced effects on the expression of PMN receptors, such as PMN CD11b and CD16, may contribute to burn-induced infection susceptibility. / Original Publication: Joakim Johansson, Florence Sjögren, Mikael Bodelsson and Folke Sjöberg, Dynamics of leukocyte receptors after severe burns: An exploratory study, 2011, BURNS, (37), 2, 227-233. http://dx.doi.org/10.1016/j.burns.2010.08.015 Copyright: Elsevier Science B.V., Amsterdam. http://www.elsevier.com/

Although platelets are anucleate, they do inherit 1500-3000 mRNA transcripts from their megakaryocyte progenitors, in addition to all the machinery essential for protein synthesis; however, there is little understanding why platelets initiate de novo synthesis of these transcripts. Our group demonstrated that fibrinogen (Fg), a ligand of platelet Glycoprotein (GP)IIb-IIIa (αIIbβ3 integrin), is required for platelet P-selectin expression and that engagement of Fg with GPIIb-IIIa is essential for this process. The present study shows that murine platelets incubated with Fg synthesize P-selectin de novo, and this synthesis is blocked by puromycin. A similar effect is also observed when platelets are incubated with fibronectin, another ligand of GPIIb-IIIa. Furthermore, platelets from both ligand- (Fg−/−, von Willebrand factor−/−, apolipoprotein A-IV−/−) and GPIIb-IIIa-deficient mice have altered proteomes. These data suggest an intricate mechanism by which engagement of platelets with their environment triggers signal-dependent translation of the platelet transcriptome, consequently altering the platelet proteome.

Although platelets are anucleate, they do inherit 1500-3000 mRNA transcripts from their megakaryocyte progenitors, in addition to all the machinery essential for protein synthesis; however, there is little understanding why platelets initiate de novo synthesis of these transcripts. Our group demonstrated that fibrinogen (Fg), a ligand of platelet Glycoprotein (GP)IIb-IIIa (αIIbβ3 integrin), is required for platelet P-selectin expression and that engagement of Fg with GPIIb-IIIa is essential for this process. The present study shows that murine platelets incubated with Fg synthesize P-selectin de novo, and this synthesis is blocked by puromycin. A similar effect is also observed when platelets are incubated with fibronectin, another ligand of GPIIb-IIIa. Furthermore, platelets from both ligand- (Fg−/−, von Willebrand factor−/−, apolipoprotein A-IV−/−) and GPIIb-IIIa-deficient mice have altered proteomes. These data suggest an intricate mechanism by which engagement of platelets with their environment triggers signal-dependent translation of the platelet transcriptome, consequently altering the platelet proteome.

The cell surface is an active area where extracellular molecules meet their receptors and affect the cellular fate by inducing for example cell proliferation and adhesion. Syndecans and integrins are two transmembrane molecules that have been suggested to fine-tune these activities, possibly in cooperation. Syndecans are proteoglycans, i.e. proteins with specific types of carbohydrate chains attached. These chains are glycosaminoglycans and either heparan sulfate (HS) or chondroitin sulfate (CS). Syndecans are known to influence cell adhesion and signaling. Integrins in turn, are important adhesion molecules that connect the extracellular matrix with the cytoskeleton, and hence can regulate cell motility. In an attempt to study how the two types of glycosaminoglycans attached to syndecan-1 can interact with integrins, a cell based model system was used and functional motility assays were performed. The results showed that HS, but not CS, on the cell surface was capable of regulating integrin-mediated cell motility. Regulation of intracellular signaling is crucial to prevent abnormal cellular behavior. In the second part of this thesis, the aim was to see how the presentation of glycosaminoglycan chains to the FGF signaling complex could affect the cellular response. When attached to the plasma membrane via syndecan-1, CS chains could support the intracellular signaling, although not promoting as strong signals as HS. When glycosaminoglycans were attached to free ectodomains of syndecan-1, both types of chains sequestered FGF2 from the receptors to the same extent, pointing towards functional overlap between CS and HS. To further study the interplay between HS and CS, their roles in the formation of pharyngeal cartilage in zebrafish were established. HS was important during chondrocyte intercalation and CS in the formation of the surrounding extracellular matrix. Further, the balance between the biosynthetic enzymes determined the ratio of HS and CS, and HS biosynthesis was prioritized over CS biosynthesis. The results presented in this thesis provide further insight into the regulation of HS biosynthesis, as well as the roles of both HS and CS on the cell surface. It is evident, that in certain situations there is a strict requirement for a certain HS structure, albeit in other situations there is a functional overlap between HS and CS.

<p>Intervertebral disc (IVD) disorders are a major contributor to disability and health costs. Disc disorders and resulting pain may be preceded by changes which first occur in the nucleus pulposus (NP) region of the IVD, where significant alterations in tissue cellularity, composition, and structure begin early in human life and continue with increasing age and degeneration. These changes coincide with the loss of a distinct cell population, notochordally-derived immature NP cells, which may play a key role in the generation and maintenance of this tissue. These cells reside in a gelatinous, highly-hydrated extracellular matrix (ECM) environment and exhibit in situ cell-matrix and cell-cell interactions which are quite distinct from cells in other regions of the disc or in other cartilagenous, including expression of laminin cell-matrix receptors and cell-associated laminin proteins. The ECM environment is known to be a key regulator of cellular behaviors, with ECM ligands and elasticity modulating cell adhesion, organization, differentiation, and phenotype. The primary motivating hypothesis of this thesis is that the unique ECM environment of the NP plays a key role in modulating immature NP cell behaviors, and that laminin ligands, in combination with ECM elasticity, modulate immature NP cell behaviors including adhesion, organization, and phenotype.</p><p>To investigate this hypothesis, flow cytometric analyses were performed to examine IVD cell integrin receptor expression over time in culture, including assessment of key laminin-binding integrin subunits. The roles of specific integrin receptors modulating NP cell adhesion to ECM proteins were identified in studies utilizing function-blocking antibodies. NP cell adhesion, spreading, and relative cell adhesion strength was assessed on various ECM constituents, including specific isoforms of laminin. Additionally, studies were performed to examine the roles of ECM ligand and substrate stiffness in modulating NP cellular organization, utilizing polyacrylamide gel substrates with tunable mechanical properties and functionalized with different ECM ligands. Finally, the role of ECM environment was examined on one key measure of NP cell function, proteoglycan production, over time in culture.</p><p>NP cells isolated from immature NP tissues were found to express high levels of the laminin-binding integrin subunit alpha 6 ex situ and maintain this expression over time in culture. Function blocking studies revealed this receptor to be a key regulator of NP cell adhesion to laminin, in contrast to cells from the adjacent AF region, which did not express this receptor nor adhere to laminin. Cell adhesion studies demonstrated that NP cells preferentially interact with two laminin isoforms, LM-511 and LM-332, in comparison to other ECM proteins, with enhanced cell attachment, spreading, and adhesion strength on surfaces coated with these ligands. These findings correspond with laminin isoform and receptor expression patterns identified in immature NP tissues. Additionally, NP cell-cell interactions were found to be modulated by both ECM ligand and substrate stiffness, with soft, laminin-functionalized substrates promoting self-assembly of NP cells into cell clusters with morphologies similar to those identified in immature NP tissues. Finally, culture of immature NP cells on soft, laminin-rich substrates was found to promote a key measure of NP cell function, proteoglycan synthesis.</p><p>The studies presented here demonstrate that immature NP cells interact uniquely with laminin extracellular matrix proteins, and that laminin ligands and matrix elasticity are two key regulators of NP cell organization and phenotype in the IVD. These findings suggest that alterations in one or both of these factors during IVD aging or degeneration may contribute to the differentiation or loss of this unique cell population. Additionally, these results indicate that soft, laminin-functionalized biomaterials may be appropriate for in vitro culture and expansion of immature NP cells, as well as for use in NP tissue engineering strategies.</p> / Dissertation

Cellular adhesion plays a crucial role in the biological function of cells, allowing them to communicate and signal, as well as physically anchor, by enabling them to adhere to either other cells or the extra cellular matrix (ECM). This process is regulated by several factors including intrinsic bond kinetics, internal cellular signaling, environment, force exerted on the bond, and force history of the bond. Concerning the force and force history dependence, the observation of catch bonds in integrin binding has asked as more questions than it has answered. To explore the force and force history dependence this process, each bond was loaded to a peak force before relaxing to a much lower force that was held for the duration of the measurement. Two different integrins were studied, both of which have in previous works exhibited a catch bond. Furthermore, the effects of different metal ion conditions and an allosteric antagonist were also studied to elucidate the conformational effects on force priming of integrin. What was observed was that I domain, or αA domain, possessing integrin, whether tested against its more active or less active binding state, changed very little in terms of off rate once the priming force was applied. However in the I domain, or αA domain, lacking integrin, the observed off rate changed as well. It seems that force priming is capable of causing integrin to bind in a stronger manner regardless of the other conditions used to either activate or inhibit binding. However the way in which the binding is strengthened depends on the receptors structure.

Epithelial to mesenchymal transition (EMT), the transdifferentation of an epithelial cell into a mesenchymal fibroblast, is a cellular process necessary for embryonic development and wound healing. However, uncontrolled EMT can result in accumulation of myofibroblasts and excessive deposition of ECM, contributing to the pathological progression of fibrotic diseases such as pulmonary fibrosis. The ability to control EMT is important for development of novel therapeutics for fibrotic pathologies and for designing novel biomaterials for tissue engineering applications seeking to promote EMT for development of complex tissues. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin-mediated interactions with extracellular matrix (ECM) proteins and soluble growth factors such as TGFβ. TGFβ, a potent inducer of EMT, is activated via cell contraction-mediated mechanical release of the growth factor from a macromolecular latency complex. Thus TGFβ activity and subsequent EMT may be influenced by the biochemical and biophysical state of the surrounding ECM. Based on these knowns, it was hypothesized that both changes in integrin engagement and increases in substrate rigidity would modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin-specific interactions with fibronectin (Fn) fragments displaying both the RGD and PHSRN binding sites facilitate cell binding through α5β1 and α3β1 integrins, and lead to maintenance of epithelial phenotype, while Fn fragments displaying only the RGD site facilitate cell binding through αv integrins and lead to EMT. An in depth investigation into α3β1 binding to Fn fragments indicates that binding is dependent on both the presence and orientation of the PHSRN site. Studies investigating the contribution of ECM stiffening on EMT responses show that increasingly rigid Fn substrates are sufficient to induce spontaneous EMT. Analysis of TGFβ-responsive genes implicate TGFβ-expression, activation or signaling as a mechanism for the observed EMT responses. Together these results suggest that the ECM micromechanical environment is a significant contributor to the onset of EMT responses and provide insights into the design of biomaterial-based microenvironments for the control of epithelial cell phenotype.

Cell adhesion to extracellular matrix (ECM) is critical to various cellular processes like cell spreading, migration, growth and apoptosis. At the tissue level, cell adhesion is important in the pathological and physiological processes that regulate the tissue morphogenesis. Cell adhesion to the ECM is primarily mediated by the integrin family of receptors. The receptors that are recruited to the surface are reinforced by structural and signaling proteins at the adhesive sites forming focal adhesions that connect the cytoskeleton to further stabilize the adhesions. The functional roles of these focal adhesions extend beyond stabilizing adhesions and transduce mechanical signals at the cell-ECM interface in various signaling events. The objective of this research is to analyze the role of the spatial distribution of the focal adhesions in stabilizing the cell adhesion to the ECM in relation to cell's internal force balance. The central hypothesis was that peripheral focal adhesions stabilize cell adhesion to ECM by providing for maximum mechanical advantage for resisting detachment as explained by the membrane peeling mechanism. Micropatterning techniques combined with robust hydrodynamic shear assay were employed to test our hypothesis. However, technical difficulties in microcontact printing stamps with small and sparse features made it challenging to analyze the role of peripheral focal adhesions in stabilizing cell adhesion. To overcome this limitation, the roof collapse phenomenon in stamps with small and sparse features (low fill factor stamps) that was detrimental to the reproduction of the adhesive geometries required to test the hypothesis was analyzed. This analysis lead to the valuable insight that the non-uniform pressure distribution during initial contact caused by parallelism error during manual microcontact printing prevented accurate replication of features on the substrate. To this end, the template of the stamp was modified so that it included an annular column around the pattern zone that acted as a collapse barrier and prevented roof collapse propagation into the pattern zone. Employing this modified stamp, the required geometries for the cell adhesion analysis were successfully reproduced on the substrates with high throughput. Adhesive areas were engineered with circular and annular patterns to discern the contribution of peripheral focal adhesions towards cell adhesion strength. The patterns were engineered such that two distinct geometries with either constant adhesive area or constant spreading area were obtained. The significance of annular patterns is that for the same total adhesive area as the circular pattern, the annular pattern provided for greater cell spreading thereby increasing the distance of the focal adhesions from the cell's center. The adhesion strength analysis was accomplished by utilizing hydrodynamic shear flow in a spinning disk device that was previously developed. The results indicate that for a constant total adhesive area, the annular patterns provide for greater adhesion strength by enhancing cell spreading area and providing for greater moment arm in resisting detachment due to shear. The next examination was the effect of the cell's internal force balance in stabilizing the cell adhesion. The working hypothesis was that microtubules provide the necessary forces to resist the tensile forces expressed by the cell contractile machinery, thereby stabilizing cell adhesion. Since microtubule disruption is known to enhance cell contractility, its effect on the cell adhesion strength was examined. Moreover, the force balance in cells was altered by engineering adhesive areas so that the cells were either spherical or completely spread and then disrupted microtubules to understand the significance of the force balance in modulating the cell adhesion strength. The results indicated that disruption of microtubules in cells on adhesive islands resulted in a 10 fold decrease in adhesion strength compared to untreated controls whereas no significant change was observed in completely spread cells between treated and untreated controls. This is in surprising contrast to the previous contractility inhibition studies which indicate a less pronounced regulation of adhesion strength for both micropatterned and spread cells. Taken together, these findings suggest that the internal force balance regulated by cell shape strongly modulates the adhesion strength though the microtubule network. In summary, this project elucidates the role of peripheral focal adhesions in regulating the cell adhesion strength. Furthermore, this study also establishes the importance of the internal force balance towards stabilizing the cell adhesion to the ECM through the microtubule network.