Biomedical subjects

Boris Hinz

Publications and source records attributed to Boris Hinz.

15 recordsLinked to original sources

PubMed2006

Isoform-specific regulation of the actin-organizing protein palladin during TGF-beta1-induced myofibroblast differentiation.

Contractile myofibroblasts are responsible for remodeling of extracellular matrix during wound healing; however, their continued activity results in various fibrocontractive diseases. Conversion of fibroblasts into myofibroblasts is induced by transforming growth factor-beta1 (TGF-beta1) and is hallmarked by the neo-expression of alpha-smooth muscle actin (alpha-SMA), a commonly used myofibroblast marker. Moreover, myofibroblast differentiation and acquisition of the contractile phenotype involves functionally important alterations in the expression of actin-organizing proteins. We investigated whether myofibroblast differentiation is accompanied by changes in the expression of palladin, a cytoskeletal protein that controls stress fiber integrity. Palladin is expressed as several isoforms, including major 3Ig (90 kDa) and 4Ig (140 kDa) forms that differ in their N-terminal sequence. Expression of the 4Ig isoform is strongly induced in fibroblast stress fibers upon TGF-beta1 treatment preceding alpha-SMA upregulation. TGF-beta1 induced upregulation of palladin is mediated both by Smad and mitogen-activated protein kinase pathways. Furthermore, palladin 4Ig-isoform is co-expressed with alpha-SMA in vivo in experimental rat wounds and in human myofibroblast-containing lesions. Taken together these results identify palladin 4Ig as a novel marker of myofibroblast conversion in vitro and in vivo. They also provide for the first time information about the signaling cascades involved in the regulation of palladin expression.

Actins↗

PubMed2006

Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers.

Expression of alpha-smooth muscle actin (alpha-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify alpha-SMA as a mechanosensitive protein that is recruited to stress fibers under high tension. Generation of this threshold tension requires the anchoring of stress fibers at sites of 8-30-microm-long "supermature" focal adhesions (suFAs), which exert a stress approximately fourfold higher (approximately 12 nN/microm2) on micropatterned deformable substrates than 2-6-microm-long classical FAs. Inhibition of suFA formation by growing myofibroblasts on substrates with a compliance of < or = 11 kPa and on rigid micropatterns of 6-microm-long classical FA islets confines alpha-SMA to the cytosol. Reincorporation of alpha-SMA into stress fibers is established by stretching 6-microm-long classical FAs to 8.1-microm-long suFA islets on extendable membranes; the same stretch producing 5.4-microm-long classical FAs from initially 4-microm-long islets is without effect. We propose that the different molecular composition and higher phosphorylation of FAs on supermature islets, compared with FAs on classical islets, accounts for higher stress resistance.

Actins↗

PubMed2005

Masters and servants of the force: the role of matrix adhesions in myofibroblast force perception and transmission.

Fibroblast-to-myofibroblast differentiation - a key event in the development of fibrocontractive diseases and in wound granulation tissue contraction - is hallmarked by the formation of stress fibers and the neo-expression of alpha-smooth muscle actin. Incorporation of the smooth muscle actin isoform into stress fibers confers to myofibroblasts a high contractile activity which is transmitted to the extracellular matrix at sites of specialized adhesions, termed 'fibronexus' in tissue and 'supermature focal adhesions' in two-dimensional cell culture. Myofibroblast differentiation requires a mechanically restrained environment in conjunction with the action of growth factors like transforming growth factor beta and specialized matrix molecules like the ED-A splice variant of fibronectin. This mini-review discusses the roles of myofibroblast adhesions in sensing matrix stress, in transmitting contractile force to the extracellular environment and in creating the high intracellular tension that is required for myofibroblast development.

Animals↗

PubMed2005

Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro.

The differentiation of fibroblasts to contractile myofibroblasts, which is characterized by de novo expression of alpha-smooth muscle actin (alpha-SMA), is crucial for wound healing and a hallmark of tissue scarring and fibrosis. These processes often follow inflammatory events, particularly in soft tissues such as skin, lung and liver. Although inflammatory cells and damaged epithelium can release transforming growth factor beta1 (TGF-beta1), which largely mediates myofibroblast differentiation, the biophysical environment of inflammation and tissue regeneration, namely increased interstitial flow owing to vessel hyperpermeability and/or angiogenesis, may also play a role. We demonstrate that low levels of interstitial (3D) flow induce fibroblast-to-myofibroblast differentiation as well as collagen alignment and fibroblast proliferation, all in the absence of exogenous mediators. These effects were associated with TGF-beta1 induction, and could be eliminated with TGF-beta1 blocking antibodies. Furthermore, alpha1beta1 integrin was seen to play an important role in the specific response to flow, as its inhibition prevented fibroblast differentiation and subsequent collagen alignment but did not block their ability to contract the gel in a separate floating gel assay. This study suggests that the biophysical environment that often precedes fibrosis, such as swelling, increased microvascular permeability and increased lymphatic drainage--all which involve interstitial fluid flow--may itself play an important role in fibrogenesis.

Cell Differentiation↗

PubMed2005

Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(-/-) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers alpha-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(-/-) mice, suggesting an impaired myofibroblast differentiation. TGF-beta signalling was clearly involved since TGF-beta1 and TGF-beta receptor type-II protein levels were decreased, while TGF-beta(1) injections into wound margins fully re-established wound closure. Since, in CD18(-/-) mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18(-/-) neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-beta1. Indeed, in neutrophil-macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-beta1 release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing.

Actins↗

PubMed2005

The N-terminal Ac-EEED sequence plays a role in alpha-smooth-muscle actin incorporation into stress fibers.

We have previously shown that the N-terminal sequence AcEEED of alpha-smooth-muscle actin causes the loss of alpha-smooth-muscle actin from stress fibers and a decrease in cell contractility when introduced in myofibroblasts as a cell-penetrating fusion peptide. Here, we have investigated the function of this sequence on stress fiber organization in living cells, using enhanced green fluorescent protein (EGFP)-tagged alpha-smooth-muscle actin. The fusion peptide provokes the gradual disappearance of EGFP fluorescence of alpha-smooth-muscle actin from stress fibers and the formation of hitherto unknown rod-like structures. In addition to alpha-smooth-muscle actin, these structures contain cytoplasmic actins, gelsolin and cofilin but not other major actin-binding proteins. These rod-like structures are also visible in wild-type fibroblasts during normal cell spreading, suggesting that they represent a physiological step in the organization of alpha-smooth-muscle actin in stress fibers. Fluorescence-recovery-after-photobleaching experiments suggest that the fusion peptide reduces the dynamics of alpha-smooth-muscle actin and its incorporation in stress fibers. Here, we propose a new mechanism of how alpha-smooth-muscle actin is incorporated in stress fibers involving the sequence Ac-EEED.

Actins↗

PubMed2004

Transgenic mice reveal novel activities of growth hormone in wound repair, angiogenesis, and myofibroblast differentiation.

An increasing number of patients are being treated with growth hormone (GH) for the enhancement of body growth but also as an anti-aging strategy. However, the side effects of GH have been poorly defined. In this study we determined the effect of GH on wound repair and its mechanisms of action at the wound site. For this purpose, we performed wound healing studies in transgenic mice overexpressing GH. Full thickness incisional and excisional wounds of transgenic animals developed extensive, highly vascularized granulation tissue. However, wound bursting strength was not increased. Wound closure was strongly delayed as a result of enhanced granulation tissue formation and impaired wound contraction. The latter effect is most likely due to a significantly reduced number of myofibroblasts at the wound site. By using in vitro studies with stressed collagen lattices, we identified GH as an inhibitor of transforming growth factor beta-induced myofibroblast differentiation, resulting in a reduction in fibroblast contractile activity. These results revealed novel roles of GH in angiogenesis and myofibroblast differentiation, which are most likely not mediated via insulin-like growth factors at the wound site. Furthermore, our data suggested that systemic GH treatment is detrimental for wound healing in healthy individuals.

Acetic Acid↗

PubMed2004

Dissecting the roles of endothelin, TGF-beta and GM-CSF on myofibroblast differentiation by keratinocytes.

Myofibroblasts are specialized fibroblasts that contribute to wound healing by producing extracellular matrix and by contracting the granulation tissue. They appear in a phase of wound healing when the dermis strongly interacts with activated epidermal keratinocytes. Direct co-culture with keratinocytes upregulates TGFbeta activity and also induces fibroblast to differentiate into alpha-smooth muscle actin (alphaSMA)-positive myofibroblasts. TGF-beta activity alone cannot completely account for alphaSMA induction in these co-cultures, and here we analyze mechanical force generation, another potent inducer of myofibroblast differentiation in this model. Using deformable silicone substrates, we show that contractile activity of fibroblasts is already induced after 1-2-days of co-culture, when fibroblasts are generally alphaSMA negative. Endothelin-1 (ET-1), the most potent inducer of smooth muscle cell contraction, was up-regulated in co-cultures, while blocking ET-1 with the ET receptor inhibitor PD156252 inhibited contraction in these early co-cultures. In 4-5 days of co-culture, however, fibroblast contractile activity correlated with an increased expression of alphaSMA expression. Stimulation of fibroblast mono-cultures with ET-1 in a low serum medium did not induce alphaSMA expression; however, ET-1 did synergize with TGF-beta. Surprisingly, GM-CSF, another mediatorstimulating myofibroblast differentiation in granulation tissue, inhibited alphaSMA expression in fibroblasts, costimulated with TGF-beta and ET-1. GM-CSF activated NFkappaB, thus interfering with TGF-beta signaling. Blocking TGFbeta and ET-1 largely impaired alphaSMA induction in co-cultures at day 7 and, in combination, almost completely prevented alphaSMA induction. Our results dissect the roles of TGF-beta and ET-1 on mechanical force generation in keratinocyte-fibroblast co-cultures, and identify GM-CSF as an inducer of myofibroblasts acting indirectly.

Antibodies, Monoclonal↗

PubMed2003

Alpha-smooth muscle actin is crucial for focal adhesion maturation in myofibroblasts.

Cultured myofibroblasts are characterized by stress fibers, containing alpha-smooth muscle actin (alpha-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of alpha-SMA-negative fibroblasts. We have investigated the role of alpha-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of alpha-SMA-negative fibroblasts. Then, myofibroblasts incorporate alpha-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When alpha-SMA expression is induced in 5 d culture by TGFbeta or low serum levels, fibroblast adhesion is further increased correlating with a "supermaturation" of FAs. Treatment of myofibroblasts with alpha-SMA fusion peptide (SMA-FP), which inhibits alpha-SMA-mediated contractile activity, reduces their adhesion to the level of alpha-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and beta3 integrin-containing FAs; alpha5 integrin distribution is not affected. FRAP of beta3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high alpha-SMA-mediated contractile activity of myofibroblast stress fibers.

Actins↗

PubMed2003

Mechanisms of force generation and transmission by myofibroblasts.

The myofibroblast has been shown to have a key role in tissue reconstruction after injury and pathological changes characterized by fibrosis. Force generation by the myofibroblast depends on the isometric contraction of stress fibers containing alpha-smooth muscle actin, and is mediated through Rho/Rho-kinase. The force is transmitted by vinculin and tensin-containing "supermature" focal adhesions, which connect stress fibers with the extracellular matrix. Force production and transmission by the myofibroblast are modulated by the coordinated action of cytokines, extracellular matrix components and mechanical tension. Regulation of these phenomena will be important for therapeutic strategies aimed at influencing fibrocontractive diseases.

Cell Differentiation↗

PubMed2003

Cell-matrix and cell-cell contacts of myofibroblasts: role in connective tissue remodeling.

It is presently accepted that fibroblast/myofibroblast modulation represents a crucial step in granulation tissue contraction and in the production of the connective tissue deformations typical of fibrocontractive diseases. In addition to synthesizing extracellular matrix (ECM) components, myofibroblasts can develop tensile force through the neoformation of alpha-smooth muscle actin (alpha-SMA) containing cytoplasmic stress fibers. Tension has been shown to be a crucial regulator of connective tissue remodeling. In order to coordinate tension distribution within connective tissue, cell-matrix and cell-cell contacts appear essential. This review addresses the formation, molecular structure and function of such structures that are characterized by their association with intracytoplasmic actin filaments. Actin associated cell-matrix adhesions appear to provide the interface between ECM components and intracellular stress fibers, thus contributing to the transmission of force to the substrate and to the detection of stress level in the matrix. Cell-cell adherens junctions appear to synchronize myofibroblast contractile activity. Further studies investigating the functions of these structures will be important for the understanding of the mechanisms of granulation tissue evolution and for the planification of strategies in view of influencing connective tissue deformations.

Actins↗

PubMed2002

The NH2-terminal peptide of alpha-smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo.

Myofibroblasts are specialized fibroblasts responsible for granulation tissue contraction and the soft tissue retractions occurring during fibrocontractive diseases. The marker of fibroblast-myofibroblast modulation is the neo expression of alpha-smooth muscle actin (alpha-SMA), the actin isoform typical of vascular smooth muscle cells that has been suggested to play an important role in myofibroblast force generation. Actin isoforms differ slightly in their NH2-terminal sequences; these conserved differences suggest different functions. When the NH2-terminal sequence of alpha-SMA Ac-EEED is delivered to cultured myofibroblast in the form of a fusion peptide (FP) with a cell penetrating sequence, it inhibits their contractile activity; moreover, upon topical administration in vivo it inhibits the contraction of rat wound granulation tissue. The NH2-terminal peptide of alpha-skeletal actin has no effect on myofibroblasts, whereas the NH2-terminal peptide of beta-cytoplasmic actin abolishes the immunofluorescence staining for this isoform without influencing alpha-SMA distribution and cell contraction. The FPs represent a new tool to better understand the specific functions of actin isoforms. Our findings support the crucial role of alpha-SMA in wound contraction. The alpha-SMA-FP will be useful for the understanding of the mechanisms of connective tissue remodeling; moreover, it furnishes the basis for a cytoskeleton-dependent preventive and/or therapeutic strategy for fibrocontractive pathological situations.

Actins↗

PubMed2002

Myofibroblasts and mechano-regulation of connective tissue remodelling.

During the past 20 years, it has become generally accepted that the modulation of fibroblastic cells towards the myofibroblastic phenotype, with acquisition of specialized contractile features, is essential for connective-tissue remodelling during normal and pathological wound healing. Yet the myofibroblast still remains one of the most enigmatic of cells, not least owing to its transient appearance in association with connective-tissue injury and to the difficulties in establishing its role in the production of tissue contracture. It is clear that our understanding of the myofibroblast its origins, functions and molecular regulation will have a profound influence on the future effectiveness not only of tissue engineering but also of regenerative medicine generally.

Actins↗

PubMedDate not supplied

Biocompatibility of bioresorbable poly(L-lactic acid) composite scaffolds obtained by supercritical gas foaming with human fetal bone cells.

The aim of this investigation was to test the biocompatibility of three-dimensional bioresorbable foams made of poly(L-lactic acid) (PLA), alone or filled with hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP), with human primary osteoblasts, using a direct contact method. Porous constructs were processed by supercritical gas foaming, after a melt-extrusion of ceramic/polymer mixture. Three neat polymer foams, with pore sizes of 170, 310, and 600 microm, and two composite foams, PLA/5 wt% HA and PLA/5 wt% beta-TCP, were examined over a 4-week culture period. The targeted application is the bone tissue-engineering field. For this purpose, human fetal and adult bone cells were chosen because of their highly osteogenic potential. The association of fetal bone cells and composite scaffold should lead to in vitro bone formation. The polymer and composite foams supported adhesion and intense proliferation of seeded cells, as revealed by scanning electron microscopy. Cell differentiation toward osteoblasts was demonstrated by alkaline phosphatase (ALP) enzymatic activity, gamma-carboxylated Gla-osteocalcin production, and the onset of mineralization. The addition of HA or beta-TCP resulted in higher ALP enzymatic activity for fetal bone cells and a stronger production of Gla-osteocalcin for adult bone cells.

Bone Substitutes↗

PubMedDate not supplied

Contraction of myofibroblasts in granulation tissue is dependent on Rho/Rho kinase/myosin light chain phosphatase activity.

During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating myofibroblasts. We examined whether regulation of myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of myofibroblasts necessary for wound closure.

Amides↗