Swelling-induced patterning in soft microchannels.

We study the effect of inflation on the swelling-induced wrinkling of thin elastic membranes in a set-up that is commonly used to create microchannels in lab-on-chip applications. Using a combination of experiments and associated numerical simulations, we demonstrate that the out-of-plane deformation of the inflated membrane and the resulting anisotropic stress lead to two distinct instabilities as the swelling progresses. The membrane first develops small-amplitude wrinkles that retain the cross-channel symmetry. Their wavelength depends on the pressure and is set in a process similar to the axisymmetric buckling of pressurised, uni-axially compressed cylindrical shells. As swelling increases, the membrane undergoes a secondary instability during which the wrinkles coarsen into large-amplitude folds whose morphology can be controlled by the degree of pre-inflation. We elucidate the fundamental mechanisms responsible for this behaviour and explain how inflation can be used as a control mechanism in the manufacture of microchannels.

Resonances in Pulsatile Channel Flow with an Elastic Wall.

Interactions between fluids and elastic solids are ubiquitous in applications ranging from aeronautical and civil engineering to physiological flows. Here we study the pulsatile flow through a two-dimensional Starling resistor as a simple model for unsteady flow in elastic vessels. We numerically solve the equations governing the flow and the large-displacement elasticity and show that the system responds as a forced harmonic oscillator with nonconventional damping. We derive an analytical prediction for the amplitude of the oscillatory wall deformation, and thus the conditions under which resonances occur or vanish.

Modulating functional connectivity between medial frontopolar cortex and amygdala by inhibitory and excitatory transcranial magnetic stimulation.

The prefrontal-limbic network in the human brain plays a major role in social cognition, especially cognitive control of emotion. The medial frontopolar cortex (mFP; Brodmann Area 10) and the amygdala are part of this network and display correlated neuronal activity in time, as measured by functional magnetic resonance imaging (fMRI). This functional connectivity is dynamic, sensitive to training, and affected in mental disorders. However, the effects of neurostimulation on functional connectivity within this network have not yet been systematically investigated. Here, we investigate the effects of both low- and high-frequency repetitive transcranial magnetic stimulation (rTMS) to the right mFP on functional connectivity between mFP and amygdala, as measured with resting state fMRI (rsfMRI). Three groups of healthy participants received either low-frequency rTMS (1 Hz; N = 18), sham TMS (1 Hz, subthreshold; N = 18) or high-frequency rTMS (20 Hz; N = 19). rsfMRI was acquired before and after (separate days). We hypothesized a modulation of functional connectivity in opposite directions compared to sham TMS through adjustment of the stimulation frequency. Groups differed in functional connectivity between mFP and amygdala after stimulation compared to before stimulation (low-frequency: decrease, high-frequency: increase). Motion or induced changes in neuronal activity were excluded as confounders. Results show that rTMS is effective for increasing and decreasing functional coherence between prefrontal and limbic regions. This finding is relevant for social and affective neuroscience as well as novel treatment approaches in psychiatry.

Curvature-Sensitive Kinesin Binding Can Explain Microtubule Ring Formation and Reveals Chaotic Dynamics in a Mathematical Model.

Microtubules are filamentous tubular protein polymers which are essential for a range of cellular behaviour, and are generally straight over micron length scales. However, in some gliding assays, where microtubules move over a carpet of molecular motors, individual microtubules can also form tight arcs or rings, even in the absence of crosslinking proteins. Understanding this phenomenon may provide important explanations for similar highly curved microtubules which can be found in nerve cells undergoing neurodegeneration. We propose a model for gliding assays where the kinesins moving the microtubules over the surface induce ring formation through differential binding, substantiated by recent findings that a mutant version of the motor protein kinesin applied in solution is able to lock-in microtubule curvature. For certain parameter regimes, our model predicts that both straight and curved microtubules can exist simultaneously as stable steady states, as has been seen experimentally. Additionally, unsteady solutions are found, where a wave of differential binding propagates down the microtubule as it glides across the surface, which can lead to chaotic motion. Whilst this model explains two-dimensional microtubule behaviour in an experimental gliding assay, it has the potential to be adapted to explain pathological curling in nerve cells.

Modeling Graphene-Polymer Heterostructure MEMS Membranes with the Föppl-von Kármán Equations.

Ultra-thin graphene-based membranes have shown significant promise for high-performance nano-electro-mechanical (NEMS) devices. The key challenge in the modeling of such membranes is that they often operate in deflection regimes where the assumptions or approximations of "pure bending" or "pure stretching" are not satisfied. We present a model of graphene-polymer heterostructure (GPH) NEMS membranes based on Föppl-von Kármán (FvK) equations which take into account both bending and stretching forces. The experimental GPH membrane shape obtained through atomic force microscopy topography mapping is compared to the inflation shapes predicted by FvK-based finite element method simulation, and they show excellent agreement with each other. When the GPH membranes are deflected under pressure in a capacitive pressure sensor configuration, the effectiveness of this model is further exemplified through accurately predicting the capacitance change of deflecting GPH membrane devices at varying pressures. This model serves as a powerful new tool in the design and development of graphene-based NEMS devices, being able to predict the performance of graphene NEMS devices or to aid in the design of device geometries to match required performances.

Adiponectin-mediated changes in effector cells involved in the pathophysiology of rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is associated with increased production of adipokines, which are cytokine-like mediators that are produced mainly in adipose tissue but also in synovial cells. Since RA synovial fibroblasts (RASFs), lymphocytes, endothelial cells, and chondrocytes are key players in the pathophysiology of RA, this study was undertaken to analyze the effects of the key adipokine adiponectin on proinflammatory and prodestructive synovial effector cells. METHODS: Lymphocytes were activated in part prior to stimulation. All cells were stimulated with adiponectin, and changes in gene and protein expression were determined by Affymetrix and protein arrays. Messenger RNA and protein levels were confirmed using semiquantitative reverse transcription-polymerase chain reaction (PCR), real-time PCR, and immunoassays. Intracellular signal transduction was evaluated using chemical signaling inhibitors. RESULTS: Adiponectin stimulation of human RASFs predominantly induced the secretion of chemokines, as well as proinflammatory cytokines, prostaglandin synthases, growth factors, and factors of bone metabolism and matrix remodeling. Lymphocytes, endothelial cells, and chondrocytes responded to adiponectin stimulation with enhanced synthesis of cytokines and various chemokines. Additionally, chondrocytes released increased amounts of matrix metalloproteinases. In RASFs, adiponectin-mediated effects were p38 MAPK and protein kinase C dependent. CONCLUSION: Our previous findings indicated that adiponectin was present in inflamed synovium, at sites of cartilage invasion, in lymphocyte infiltrates, and in perivascular areas. The findings of the present study indicate that adiponectin induces gene expression and protein synthesis in human RASFs, lymphocytes, endothelial cells, and chondrocytes, supporting the concept of adiponectin being involved in the pathophysiologic modulation of RA effector cells. Adiponectin promotes inflammation through cytokine synthesis, attraction of inflammatory cells to the synovium, and recruitment of prodestructive cells via chemokines, thus promoting matrix destruction at sites of cartilage invasion.

Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis.

Bmx/Etk non-receptor tyrosine protein kinase has been implicated in endothelial cell migration and tube formation in vitro. However, the role of Bmx in vivo is not known. Bmx is highly induced in the vasculature of ischemic hind limbs. We used both mice with a genetic deletion of Bmx (Bmx-KO mice) and transgenic mice expressing a constitutively active form of Bmx under the endothelial Tie-2 enhancer/promoter (Bmx-SK-Tg mice) to study the role of Bmx in ischemia-mediated arteriogenesis/angiogenesis. In response to ischemia, Bmx-KO mice had markedly reduced, whereas Bmx-SK-Tg mice had enhanced, clinical recovery, limb perfusion, and ischemic reserve capacity when compared with nontransgenic control mice. The functional outcomes in these mice were correlated with ischemia-initiated arteriogenesis, capillary formation, and vessel maturation as well as Bmx-dependent expression/activation of TNF receptor 2- and VEGFR2-mediated (TNFR2/VEGFR2-mediated) angiogenic signaling in both hind limb and bone marrow. More importantly, results of bone marrow transplantation studies showed that Bmx in bone marrow-derived cells plays a critical role in the early phase of ischemic tissue remodeling. Our study provides the first demonstration to our knowledge that Bmx in endothelium and bone marrow plays a critical role in arteriogenesis/angiogenesis in vivo and suggests that Bmx may be a novel target for the treatment of vascular diseases such as coronary artery disease and peripheral arterial disease.

Angiopoietin-2 impairs revascularization after limb ischemia.

Angiopoietins play important roles in the formation of neovessels and complex vascular networks. Angiopoietin (Ang)-1 and Ang-2 belong to a family of growth factors that display opposing effects on the activation of Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2). Endothelial Ang-2 expression is associated with vessel destabilization and regulates a balance between vascular regression and growth. To elucidate, in particular, the role of Ang-2 after arterial artery occlusion in the mouse limb, we applied a transgenic animal model with targeted Ang-2 expression in endothelial cells. We show here that restoration of blood flow in Ang-2:Tie1 transgenic mice is dramatically impaired when Ang-2 expression is induced in the vasculature. The defective restoration of perfusion in Ang-2 transgenic mice is evidenced by reduced collateral artery growth, which typically occurs to compensate for flow deficits after occlusion of the large conductance artery. Furthermore, reduced movement capacities and higher incidents of necrosis are consequently observed in the transgenic limbs as compared with controls. Mechanistically, the observed effects are attributed to defective smooth muscle cell recruitment in Ang-2 transgenic mice. Moreover, distinct Ang-2 levels in the genetically modified animals clearly correlated with the magnitude of reduced perfusion. In conclusion, our studies define Ang-2 as an important molecule for the progression of collateral artery growth and angiogenesis during ischemia and suggest precise Ang-2 dosage activities to accomplish blood vessel growth.

The range of adaptation by collateral vessels after femoral artery occlusion.

Natural adaptation to femoral artery occlusion in animals by collateral artery growth restores only approximately 35% of adenosine-recruitable maximal conductance (C(max)) probably because initially elevated fluid shear stress (FSS) quickly normalizes. We tested the hypothesis whether this deficit can be mended by artificially increasing FSS or whether anatomical restraints prevent complete restitution. We chronically increased FSS by draining the collateral flow directly into the venous system by a side-to-side anastomosis between the distal stump of the occluded femoral artery and the accompanying vein. After reclosure of the shunt collateral flow was measured at maximal vasodilatation. C(max) reached 100% already at day 7 and had, after 4 weeks, surpassed (2-fold) the C(max) of the normal vasculature before occlusion. Expression profiling showed upregulation of members of the Rho-pathway (RhoA, cofilin, focal adhesion kinase, vimentin) and the Rho-antagonist Fasudil markedly inhibited arteriogenesis. The activities of Ras and ERK-1,-2 were markedly increased in collateral vessels of the shunt experiment, and infusions of L-NAME and L-NNA strongly inhibited MAPK activity as well as shunt-induced arteriogenesis. Infusions of the peroxinitrite donor Sin-1 inhibited arteriogenesis. The radical scavengers urate, ebselen, SOD, and catalase had no effect. We conclude that increased FSS can overcome the anatomical restrictions of collateral arteries and is potentially able to completely restore maximal collateral conductance. Increased FSS activates the Ras-ERK-, the Rho-, and the NO- (but not the Akt-) pathway enabling collateral artery growth.

Endothelial nitric oxide synthase activity is essential for vasodilation during blood flow recovery but not for arteriogenesis.

OBJECTIVE: Arteriogenesis is the major mechanism of vascular growth, which is able to compensate for blood flow deficiency after arterial occlusion. Endothelial nitric oxide synthase (eNOS) activity is essential for neovascularization, however its specific role in arteriogenesis remains unclear. We studied the role of eNOS in arteriogenesis using 3 mouse strains with different eNOS expression. METHODS AND RESULTS: Distal femoral artery ligation was performed in eNOS-overexpressing mice (eNOStg), eNOS-deficient (eNOS-/-) mice, and wild type (WT) controls. Tissue perfusion and collateral-dependent blood flow were significantly increased in eNOStg mice compared with WT only immediately after ligation. In eNOS-/- mice, although tissue perfusion remained significantly decreased, collateral-dependent blood flow was only decreased until day 7, suggesting normal, perhaps delayed collateral growth. Histology confirmed no differences in collateral arteries of eNOStg, eNOS-/-, and WT mice at 1 and 3 weeks. Administration of an NO donor induced vasodilation in collateral arteries of eNOS-/- mice, but not in WT, identifying the inability to vasodilate collateral arteries as main cause of impaired blood flow recovery in eNOS-/- mice. CONCLUSIONS: This study demonstrates that eNOS activity is crucial for NO-mediated vasodilation of peripheral collateral vessels after arterial occlusion but not for collateral artery growth.

The mechanics of airway closure.

We describe how surface-tension-driven instabilities of the lung's liquid lining may lead to pulmonary airway closure via the formation of liquid bridges that occlude the airway lumen. Using simple theoretical models, we demonstrate that this process may occur via a purely fluid-mechanical "film collapse" or through a coupled, fluid-elastic "compliant collapse" mechanism. Both mechanisms can lead to airway closure in times comparable with the breathing cycle, suggesting that surface tension is the primary mechanical effect responsible for the closure observed in peripheral regions of the human lungs. We conclude by discussing the influence of additional effects not included in the simple models, such as gravity, the presence of pulmonary surfactant, respiratory flow and wall motion, the airways' geometry, and the mechanical structure of the airway walls.

Expression of PSACH-associated mutant COMP in tendon fibroblasts leads to increased apoptotic cell death irrespective of the secretory characteristics of mutant COMP.

OBJECTIVE: Pseudoachondroplasia (PSACH) is a dominantly inherited chondrodysplasia associated with mutations of cartilage oligomeric matrix protein (COMP), characterized clinically by disproportionate dwarfism and laxity of joints and ligaments. Studies in chondrocytes and cartilage biopsies suggest that the cartilage disease is caused by retention of mutant COMP in the endoplasmic reticulum of chondrocytes and by disruption of the collagen network of the extracellular matrix. The pathogenesis of the tendon disease remains unclear in the absence of a cell culture model, with available tendon biopsies leading to conflicting results with respect to the intracellular retention of mutant COMP. METHODS: We established a cell culture model using adenoviral gene transfer in tendon fibroblast cultures. We compared the effect of expression of three PSACH-associated COMP mutants and the wildtype protein on COMP secretion, matrix composition and cellular viability. RESULTS: Our results show that mutants D475N and D469Delta are retained within the endoplasmic reticulum of tendon cells similar to what is known from chondrocytes, whereas mutant H587R is secreted like wildtype COMP. In spite of this difference, the collagen I matrix formed in culture appears disturbed for all three mutants. All COMP-mutants induce apoptotic cell death irrespective of their differing secretion patterns. CONCLUSION: Pathogenic pathways leading to tendon disease in humans appear to be heterogeneous between different COMP mutants.

Impact of mouse strain differences in innate hindlimb collateral vasculature.

OBJECTIVE: To assess the importance of genetic background for collateral artery development. METHODS AND RESULTS: C57BL/6, BALB/c and 129S2/Sv mice were studied after femoral artery ligation by laser Doppler imaging, visible light oximetry, time-of-flight-magnetic resonance imaging, and treadmill testing; C57BL/6 and BALB/c also underwent electron paramagnetic resonance (EPR) oximetry, x-ray angiography, and histology. C57BL/6 had the least initial distal ischemia and most complete recovery. BALB/c had the most severe initial ischemia and poorest recovery. BALB/c had some vasodilatory reserve in their ligated limbs not seen in the other strains at 3 weeks. By in vivo TOF-magnetic resonance angiography, C57BL/6 had larger preexistent and developed collaterals. By x-ray angiography, C57BL/6 had a higher collateral-dependent filling score and number of visible collaterals immediately after femoral ligation and a higher number of visible collaterals at 1 week but not at 4 weeks. EPR oximetry and histology revealed hypoxia and tissue damage in regions of collateral growth of BALB/c but not C57BL/6 mice. In C57BL/6 BrdUrd uptake in the thigh was limited to larger vessels and isolated perivascular cells. Proliferative activity in collateral arterioles was similar in both strains. CONCLUSIONS: Genetic differences in preexistent collateral vasculature can profoundly affect outcome and milieu for compensatory collateral artery growth after femoral artery occlusion.

Influence of mechanical, cellular, and molecular factors on collateral artery growth (arteriogenesis).

Growth of collateral blood vessels (arteriogenesis) is potentially able to preserve structure and function of limbs and organs after occlusion of a major artery. The success of the remodeling process depends on the following conditions: (1) existence of an arteriolar network that connects the preocclusive with the postocclusive microcirculation; (2) activation of the arteriolar endothelium by elevated fluid shear stress; (3) invasion (but not incorporation) of bone marrow-derived cells; and (4) proliferation of endothelial and smooth muscle cells. Most organs of most mammals including man can rely on the existence of interconnecting arterioles in most organs and tissues with heart being the exception in rodents and pigs. Arterial occlusion lowers the pressure in the distal vasculature thereby creating a pressure gradient favoring increased flow through preexisting collaterals. This increases fluid shear stress leading to endothelial activation with cellular edema, upregulation of adhesion molecules, mitogenic-, thrombogenic-, and fibrinolytic factors, leading to monocyte invasion with matrix digestion. Smooth muscle cells migrate and proliferate and the vessel enlarges under the influence of increasing circumferential wall stress. Growth factors involved belong to the FGF family and signaling proceeds via the Ras/Raf- and the Rho cascades. Increases in vascular radius and wall thickness restore fluid shear stress and circumferential wall stress to normal levels and growth stops. Although increases in collateral vessel size are very substantial their maximal conductance amounts to only 40% of normal. Forced increases in FSS can reach almost 100%.

Involvement of the fibroblast growth factor system in adaptive and chemokine-induced arteriogenesis.

Fibroblast growth factors (FGFs) have been applied in a variety of therapeutic and experimental studies to improve collateral blood flow. However, the pathophysiological role and the temporospatial expression of the FGFs and their receptors during arteriogenesis have never been elucidated in vivo. Here, we report that collateral artery growth in its early phase is associated with an increased expression of FGF receptor-1 (FGFR-1) and syndecan-4 on mRNA and protein levels as well as with an increased kinase activity of FGFR-1 in a rabbit model of arteriogenesis. However, the mRNA levels of FGF-1 and -2 remained constant. Our data suggest that these growth factors are supplied by endothelial attracted monocytes that, in turn, produce and deliver the FGFs to growing collateral arteries. Monocyte chemoattractant protein-1-stimulated arteriogenesis was strongly reduced in rabbits by application of the FGF inhibitor polyanetholesulfonic acid, indicating that the monocyte-related arteriogenesis (as well as the unstimulated adaptation proper) is promoted by FGFs. In summary, this study shows that arteriogenesis is associated with an increased expression of the FGFRs at the site of the vessel, whereas the growth-promoting ligands are supplied by monocytes in a paracrine way.

Bone marrow-derived cells do not incorporate into the adult growing vasculature.

Bone marrow-Derived cells have been proposed to form new vessels or at least incorporate into growing vessels in adult organisms under certain physiological and pathological conditions. We investigated whether bone marrow-Derived cells incorporate into vessels using mouse models of hindlimb ischemia (arteriogenesis and angiogenesis) and tumor growth. C57BL/6 wild-type mice were lethally irradiated and transplanted with bone marrow cells from littermates expressing enhanced green fluorescent protein (GFP). At least 6 weeks after bone marrow transplantation, the animals underwent unilateral femoral artery occlusions with or without pretreatment with vascular endothelial growth factor or were subcutaneously implanted with methylcholanthrene-induced fibrosarcoma (BFS-1) cells. Seven and 21 days after surgery, proximal hindlimb muscles with growing collateral arteries and ischemic gastrocnemius muscles as well as grown tumors and various organs were excised for histological analysis. We failed to colocalize GFP signals with endothelial or smooth muscle cell markers. Occasionally, the use of high-power laser scanning confocal microscopy uncovered false-positive results because of overlap of different fluorescent signals from adjacent cells. Nevertheless, we observed accumulations of GFP-positive cells around growing collateral arteries (3-fold increase versus nonoccluded side, P<0.001) and in ischemic distal hindlimbs. These cells were identified as fibroblasts, pericytes, and primarily leukocytes that stained positive for several growth factors and chemokines. Our findings suggest that in the adult organism, bone marrow-Derived cells do not promote vascular growth by incorporating into vessel walls but may function as supporting cells.

VEGFR-1-selective VEGF homologue PlGF is arteriogenic: evidence for a monocyte-mediated mechanism.

Two signaling receptors for vascular endothelial growth factor (VEGF) in the vasculature are known with not yet well-understood roles in collateral vessel growth (arteriogenesis). In this study, we examined the involvement of the two VEGF receptors in arteriogenesis. Therefore, we used the VEGF homologue placenta growth factor (PlGF), which only binds to VEGFR-1 and VEGF-E, which only recognizes VEGFR-2. These peptides were locally infused over 7 days after ligation of the femoral artery in the rabbit. Evaluation of collateral growth by determining collateral conductance and angiographic scores demonstrated that the VEGFR-1-specific PlGF contributed significantly more to arteriogenesis than the VEGFR-2 specific VEGF-E. The combination of VEGF-E and PlGF did not exceed the effect of PlGF alone, indicating that cooperation of the two VEGF receptors in endothelial cell signaling is not required for arteriogenesis. In an in vitro model of angiogenesis, VEGF and VEGF-E were comparably active, whereas PlGF displayed no activity when given alone and did not further increase the effects of VEGF or VEGF-E. However, PlGF was as potent as VEGF when monocyte activation was assessed by monitoring integrin surface expression. In addition, accumulation of activated monocytes/macrophages in the periphery of collateral vessels in PlGF-treated animals was observed. Furthermore, in monocyte-depleted animals, the ability of PlGF to enhance collateral growth in the rabbit model and to rescue impaired arteriogenesis in PlGF gene-deficient mice was abrogated. Together, these data indicate that the arteriogenic activity observed with the VEGFR-1-specific PlGF is caused by its monocyte-activating properties.

Collateral artery growth (arteriogenesis) after experimental arterial occlusion is impaired in mice lacking CC-chemokine receptor-2.

Arteriogenesis has been associated with the presence of monocytes/macrophages within the collateral vessel wall. Induced macrophage migration in vivo is driven by the binding of monocyte chemoattractant protein-1 (MCP-1, or CCL2 in the new nomenclature) to the CCR2-chemokine receptor on macrophages. To determine whether the CCL2-CCR2 signaling pathway is involved in the accumulation of macrophages in growing collateral vessels, we used mice that are deficient in CCR2 in a model of experimental arterial occlusion and collateral vessel growth. In an in vitro CCL2-driven chemotaxis assay, mononuclear cells isolated from wild-type BALB/c mice exhibited CCL2 concentration-dependent migration, whereas this migration was abolished in cells from CCR2(-/-) mice on a BALB/c genetic background. In vivo, blood flow recovery as measured by laser Doppler (LDI) and MRI (MRI) was impaired in CCR2(-/-) mice on either the BALB/c or C57BL/6 genetic backgrounds. Three weeks after femoral artery ligation, LDI perfusion ratio of operated versus nonoperated distal hindlimb in BALB/c wild-type mice increased to 0.45+/-0.06 and in CCR2(-/-) animals only to 0.21+/-0.03 (P<0.01). In C57BL/6 mice, ratio increased to 0.96+/-0.09 and 0.85+/-0.08 (P<0.05), respectively. MRI at 3 weeks (0.76+/-0.06 versus 0.62+/-0.01; P<0.05) and hemoglobin oxygen saturation measurements confirmed these findings. Active foot movement score significantly decreased and gastrocnemius muscle atrophy was significantly greater in CCR2(-/-) mice. Morphometric analysis showed a lesser increase in collateral vessel diameters in CCR2(-/-) mice. Importantly, the number of invaded monocytes/macrophages in the perivascular space of collateral arteries of CCR2(-/-) animals was dramatically reduced in comparison to wild-type mice. In conclusion, our results demonstrate that the CCR2 signaling pathway is essential for efficient collateral artery growth.

Circulating vascular progenitor cells do not contribute to compensatory lung growth.

The biological principles that underlie the induction and process of alveolization in the lung as well as the maintenance of the complex lung tissue structure are one of the major obstacles in pulmonary medicine today. Bone marrow-derived cells have been shown to participate in angiogenesis, vascular repair, and remodeling of various organs. We addressed this phenomenon in the lung vasculature of mice in a model of regenerative lung growth. C57BL/6 mice were transplanted with bone marrow from one of three different reporter gene-transgenic strains. flk-1+/lacZ mice, tie-2/lacZ transgenic mice (both exhibiting endothelial cell-specific reporter gene expression), and ubiquitously enhanced green fluorescent protein (eGFP)-expressing mice served as marrow donors. After hematopoietic recovery, compensatory lung growth was induced by unilateral pneumonectomy and led to complete restoration of initial lung volume and surface area. The lungs were consecutively investigated for bone marrow-derived vascular cells by lacZ staining and immunohistochemistry for phenotype identification of vascular cells. lacZ- or eGFP-expressing bone marrow-derived endothelial cells could not be found in microvascular regions of alveolar septa. Single eGFP-positive endothelial cells were detected in pulmonary arteries at very low frequencies, whereas no eGFP-positive vascular smooth muscle cells were observed. In conclusion, we demonstrate in a model of lung growth and alveolization in adult mice the absence of significant bone marrow-derived progenitor cell contribution to the concomitant vascular growth and remodeling processes.

Mechanosensitive p27Kip1 regulation and cell cycle entry in vascular smooth muscle cells.

BACKGROUND: Cyclic stretch plays an important role in the homeostasis of vessel structure. Increased forces might, however, contribute to remodeling processes, resulting in vascular proliferative diseases. The initial molecular events necessary for mechanosensitive cell cycle entry of quiescent smooth muscle cells are poorly understood. METHODS AND RESULTS: In this study, we demonstrate that mechanical strain resulted in a rapid, integrin-dependent but mitogen-independent activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) in quiescent vascular smooth muscle cells. Subsequently, downstream ALL 1 fused gene from chromosome X (AFX)-like forkhead transcription factors were inactivated, leading to transcriptional downregulation of p27Kip1. This contrasted with the posttranscriptional protein reduction of p27Kip1 in cells stimulated with serum mitogens. Stretch-mediated p27Kip1 downregulation was accompanied by activation of cyclin-dependent kinase 2, hyperphosphorylation of retinoblastoma protein, and proliferation. Forkhead transcription factor inactivation and p27Kip1 downregulation were prevented by the PI3-K inhibitors wortmannin and LY294002. Pharmacological blockade of other kinases, such as p42/44, p38, and protein kinase A or C, did not influence the mechanosensitive gene regulation. p27Kip1 downregulation and cell cycle entry were, however, prevented by overexpression of a constitutively inactive form of Akt or constitutively active forms of forkhead transcription factors. CONCLUSIONS: Our data demonstrate that the earliest cell cycle events can occur in a solely mechanosensitive fashion. Vascular smooth muscle cells are, furthermore, able to use transcriptional or posttranscriptional mechanisms to regulate p27Kip1, depending on the stimulus to which they are exposed. This observation has novel implications for understanding of vascular proliferative diseases.