Pro-atherosclerotic disturbed flow disrupts caveolin-1 expression, localization, and function via glycocalyx degradation.

BACKGROUND: Endothelial-dependent atherosclerosis develops in a non-random pattern in regions of vessel bending and bifurcations, where blood flow exhibits disturbed flow (DF) patterns. In contrast, uniform flow (UF), normal endothelium, and healthy vessel walls co-exist within straight vessels. In clarifying how flow protectively or atherogenically regulates endothelial cell behavior, involvement of the endothelial surface glycocalyx has been suggested due to reduced expression in regions of atherosclerosis development. Here, we hypothesized that pro-atherosclerotic endothelial dysfunction occurs as a result of DF-induced reduction in glycocalyx expression and subsequently impairs endothelial sensitivity to flow. Specifically, we propose that glycocalyx degradation can induce pro-atherosclerotic endothelial dysfunction through decreased caveolin-1 and endothelial nitric oxide synthase expression and localization. METHODS: We studied endothelial cells in atherosclerotic-prone DF and atherosclerotic-resistant UF conditions in parallel plate flow culture and in C57Bl/6 mice. The effects of flow conditioning on endothelial cell behavior were quantified using immunocytochemistry. The glycocalyx was fluorescently labeled for wheat germ agglutinin, which serves as a general glycocalyx label, and heparan sulfate, a major glycocalyx component. Additionally, mechanosensitivity was assessed by immunocytochemical fluorescence expression and function of caveolin-1, the protein that forms the mechanosignaling caveolar invaginations on the endothelial surface, total endothelial-type nitric oxide synthase (eNOS), which synthesizes nitric oxide, and serine 1177 phosphorylated eNOS (eNOS-pS1177), which is the active form of eNOS. Caveolin function and eNOS expression and activation were correlated to glycocalyx expression. Heparinase III enzyme was used to degrade a major glycocalyx component, HS, to identify the role of the glycocalyx in caveoin-1 and eNOS-pS1177 regulation. RESULTS: Results confirmed that DF reduces caveolin-1 expression and abolishes most of its subcellular localization preferences, when compared to the effect of UF. DF down-regulates caveolin-1 mechanosignaling, as indicated by its reduced colocalization with serine 1177 phosphorylated endothelial-type nitric oxide synthase (eNOS-pS1177), a vasoregulatory signaling molecule whose activity is regulated by its residence in caveolae. As expected, DF inhibited glycocalyx expression compared to UF. In the absence of heparan sulfate, a major glycocalyx component, UF-conditioned endothelial cells exhibited near DF-like caveolin-1 expression, localization, and colocalization with eNOS-pS1177. CONCLUSIONS: This is the first demonstration of a flow-defined role of the glycocalyx in caveolae expression and function related to vasculoprotective endothelial mechanosensitivity that defends against atherosclerosis. The results suggest that a glycocalyx-based therapeutic targeted to areas of atherosclerosis development could prevent disease initiation and progression.

Matrix- and plasma-derived peptides promote tissue-specific injury responses and wound healing in diabetic swine.

BACKGROUND: Non-healing wounds are a major global health concern and account for the majority of non-traumatic limb amputations worldwide. However, compared to standard care practices, few advanced therapeutics effectively resolve these injuries stemming from cardiovascular disease, aging, and diabetes-related vasculopathies. While matrix turnover is disrupted in these injuries, debriding enzymes may promote healing by releasing matrix fragments that induce cell migration, proliferation, and morphogenesis, and plasma products may also stimulate these processes. Thus, we created matrix- and plasma-derived peptides, Comb1 and UN3, which induce cellular injury responses in vitro, and accelerate healing in rodent models of non-healing wounds. However, the effects of these peptides in non-healing wounds in diabetes are not known. Here, we interrogated whether these peptides stimulate healing in a diabetic porcine model highly reminiscent of human healing impairments in type 1 and type 2-diabetes. METHODS: After 3-6 weeks of streptozotocin-induced diabetes, full-thickness wounds were surgically created on the backs of adult female Yorkshire swine under general anesthesia. Comb1 and UN3 peptides or sterile saline (negative control) were administered to wounds daily for 3-7 days. Following sacrifice, wound tissues were harvested, and quantitative histological and immunohistochemical analyses were performed for wound closure, angiogenesis and granulation tissue deposition, along with quantitative molecular analyses of factors critical for angiogenesis, epithelialization, and dermal matrix remodeling. RESULTS: Comb1 and UN3 significantly increase re-epithelialization and angiogenesis in diabetic porcine wounds, compared to saline-treated controls. Additionally, fluorescein-conjugated Comb1 labels keratinocytes, fibroblasts, and vascular endothelial cells in porcine wounds, and Far western blotting reveals these cell populations express multiple fluorescein-Comb1-interacting proteins in vitro. Further, peptide treatment increases mRNA expression of several pro-angiogenic, epithelializing, and matrix-remodeling factors, importantly including balanced inductions in matrix metalloproteinase-2, -9, and tissue inhibitor of metalloproteinases-1, lending further insight into their mechanisms. CONCLUSIONS: Comb1 and UN3 stimulate wound resolution in diabetic Yorkshire swine through upregulation of multiple reparative growth factors and cytokines, especially matrix metalloproteinases and inhibitors that may aid in reversing the proteolytic imbalance characteristic of chronically inflamed non-healing wounds. Together, these peptides should have great therapeutic potential for all patients in need of healing, regardless of injury etiology.

Pericyte contractility controls endothelial cell cycle progression and sprouting: insights into angiogenic switch mechanics.

Microvascular stability and regulation of capillary tonus are regulated by pericytes and their interactions with endothelial cells (EC). While the RhoA/Rho kinase (ROCK) pathway has been implicated in modulation of pericyte contractility, in part via regulation of the myosin light chain phosphatase (MLCP), the mechanisms linking Rho GTPase activity with actomyosin-based contraction and the cytoskeleton are equivocal. Recently, the myosin phosphatase-RhoA-interacting protein (MRIP) was shown to mediate the RhoA/ROCK-directed MLCP inactivation in vascular smooth muscle. Here we report that MRIP directly interacts with the ß-actin-specific capping protein ßcap73. Furthermore, manipulation of MRIP expression influences pericyte contractility, with MRIP silencing inducing cytoskeletal remodeling and cellular hypertrophy. MRIP knockdown induces a repositioning of ßcap73 from the leading edge to stress fibers; thus MRIP-silenced pericytes increase F-actin-driven cell spreading twofold. These hypertrophied and cytoskeleton-enriched pericytes demonstrate a 2.2-fold increase in contractility upon MRIP knockdown when cells are plated on a deformable substrate. In turn, silencing pericyte MRIP significantly affects EC cycle progression and angiogenic activation. When MRIP-silenced pericytes are cocultured with capillary EC, there is a 2.0-fold increase in EC cycle entry. Furthermore, in three-dimensional models of injury and repair, silencing pericyte MRIP results in a 1.6-fold elevation of total tube area due to EC network formation and increased angiogenic sprouting. The pivotal role of MRIP expression in governing pericyte contractile phenotype and endothelial growth should lend important new insights into how chemomechanical signaling pathways control the "angiogenic switch" and pathological angiogenic induction.

Targeted gene inactivation of calpain-1 suppresses cortical degeneration due to traumatic brain injury and neuronal apoptosis induced by oxidative stress.

Calpains are calcium-regulated cysteine proteases that have been implicated in the regulation of cell death pathways. Here, we used our calpain-1 null mouse model to evaluate the function of calpain-1 in neural degeneration following a rodent model of traumatic brain injury. In vivo, calpain-1 null mice show significantly less neural degeneration and apoptosis and a smaller contusion 3 days post-injury than wild type littermates. Protection from traumatic brain injury corroborated with the resistance of calpain-1 neurons to apoptosis induced by oxidative stress. Biochemical analysis revealed that caspase-3 activation, extracellular calcium entry, mitochondrial membrane permeability, and release of apoptosis-inducing factor from mitochondria are partially blocked in the calpain-1 null neurons. These findings suggest that the calpain-1 knock-out mice may serve as a useful model system for neuronal protection and apoptosis in traumatic brain injury and other neurodegenerative disorders in which oxidative stress plays a role.

JAGGED1 signaling regulates hemangioma stem cell-to-pericyte/vascular smooth muscle cell differentiation.

OBJECTIVE: The aim of our study is to determine the cellular and molecular origin for the pericytes in infantile hemangioma (IH) and their functional role in the formation of pathological blood vessels. METHODS AND RESULTS: Here we show that IH-derived stem cells (HemSCs) form pericyte-like cells. With in vitro studies, we demonstrate that HemSC-to-pericyte differentiation depends on direct contact with endothelial cells. JAGGED1 expressed ectopically in fibroblasts was sufficient to induce HemSCs to acquire a pericyte-like phenotype, indicating a critical role for JAGGED1. In vivo, we blocked pericyte differentiation with recombinant JAGGED1, and we observed reduced formation of blood vessels, with an evident lack of pericytes. Silencing JAGGED1 in the endothelial cells reduced blood vessel formation and resulted in a paucity of pericytes. CONCLUSIONS: Our data show that endothelial JAGGED1 controls HemSC-to-pericyte differentiation in a murine model of IH and suggests that pericytes have a fundamental role in formation of blood vessels in IH.

Acute and impaired wound healing: pathophysiology and current methods for drug delivery, part 1: normal and chronic wounds: biology, causes, and approaches to care.

This is the first installment of 2 articles that discuss the biology and pathophysiology of wound healing, review the role that growth factors play in this process, and describe current ways of growth factor delivery into the wound bed. Part 1 discusses the latest advances in clinicians' understanding of the control points that regulate wound healing. Importantly, biological similarities and differences between acute and chronic wounds are considered, including the signaling pathways that initiate cellular and tissue responses after injury, which may be impeded during chronic wound healing.

Acute and impaired wound healing: pathophysiology and current methods for drug delivery, part 2: role of growth factors in normal and pathological wound healing: therapeutic potential and methods of delivery.

This is the second of 2 articles that discuss the biology and pathophysiology of wound healing, reviewing the role that growth factors play in this process and describing the current methods for growth factor delivery into the wound bed.

Developing a transwell millifluidic device for studying blood-brain barrier endothelium.

Blood-brain barrier (BBB) endothelial cell (EC) function depends on flow conditions and on supportive cells, like pericytes and astrocytes, which have been shown to be both beneficial and detrimental for brain EC function. Most studies investigating BBB EC function lack physiological relevance, using sub-physiological shear stress magnitudes and/or omitting pericytes and astrocytes. In this study, we developed a millifluidic device compatible with standard transwell inserts to investigate BBB function. In contrast to standard polydimethylsiloxane (PDMS) microfluidic devices, this model allows for easy, reproducible shear stress exposure without common limitations of PDMS devices such as inadequate nutrient diffusion and air bubble formation. In no-flow conditions, we first used the device to examine the impact of primary human pericytes and astrocytes on human brain microvascular EC (HBMEC) barrier integrity. Astrocytes, pericytes, and a 1-to-1 ratio of both cell types increased HBMEC barrier integrity via reduced 3 and 40 kDa fluorescent dextran permeability and increased claudin-5 expression. There were differing levels of low 3 kDa permeability in HBMEC-pericyte, HBMEC-astrocyte, and HBMEC-astrocyte-pericyte co-cultures, while levels of low 40 kDa permeability were consistent across co-cultures. The 3 kDa findings suggest that pericytes provide more barrier support to the BBB model compared to astrocytes, although both supportive cell types are permeability reducers. Incorporation of 24-hour 12 dynes per cm2 flow significantly reduced dextran permeability in HBMEC monolayers, but not in the tri-culture model. These results indicate that tri-culture may exert more pronounced impact on overall BBB permeability than flow exposure. In both cases, monolayer and tri-culture, flow exposure interestingly reduced HBMEC expression of both claudin-5 and occludin. ZO-1 expression, and localization at cell-cell junctions increased in the tri-culture but exhibited no apparent change in the HBMEC monolayer. Under flow conditions, we also observed HBMEC alignment in the tri-culture but not in HBMEC monolayers, indicating supportive cells and flow are both essential to observe brain EC alignment in vitro. Collectively, these results support the necessity of physiologically relevant, multicellular BBB models when investigating BBB EC function. Consideration of the roles of shear stress and supportive cells within the BBB is critical for elucidating the physiology of the neurovascular unit.

Advanced Wound Diagnostics: Toward Transforming Wound Care into Precision Medicine.

Significance: Nonhealing wounds are an ever-growing global pandemic, with mortality rates and management costs exceeding many common cancers. Although our understanding of the molecular and cellular factors driving wound healing continues to grow, standards for diagnosing and evaluating wounds remain largely subjective and experiential, whereas therapeutic strategies fail to consistently achieve closure and clinicians are challenged to deliver individualized care protocols. There is a need to apply precision medicine practices to wound care by developing evidence-based approaches, which are predictive, prescriptive, and personalized. Recent Advances: Recent developments in "advanced" wound diagnostics, namely biomarkers (proteases, acute phase reactants, volatile emissions, and more) and imaging systems (ultrasound, autofluorescence, spectral imaging, and optical coherence tomography), have begun to revolutionize our understanding of the molecular wound landscape and usher in a modern age of therapeutic strategies. Herein, biomarkers and imaging systems with the greatest evidence to support their potential clinical utility are reviewed. Critical Issues: Although many potential biomarkers have been identified and several imaging systems have been or are being developed, more high-quality randomized controlled trials are necessary to elucidate the currently questionable role that these tools are playing in altering healing dynamics or predicting wound closure within the clinical setting. Future Directions: The literature supports the need for the development of effective point-of-care wound assessment tools, such as a platform diagnostic array that is capable of measuring multiple biomarkers at once. These, along with advances in telemedicine, synthetic biology, and "smart" wearables, will pave the way for the transformation of wound care into a precision medicine. Clinical Trial Registration number: NCT03148977.

Scaffold proteins IRSp53 and spinophilin regulate localized Rac activation by T-lymphocyte invasion and metastasis protein 1 (TIAM1).

The Rac exchange factor Tiam1 is involved in diverse cell functions and signaling pathways through multiple protein interactions, raising the question of how signaling and functional specificity are achieved. We have shown that Tiam1 interactions with different scaffold proteins activate different Rac-dependent pathways by recruiting specific Rac effector proteins, and reasoned that there must be regulatory mechanisms governing each interaction. Fibroblasts express at least two Tiam1-interacting proteins, insulin receptor substrate protein 53 kDa (IRSp53) and spinophilin. We used fluorescent resonance energy transfer (FRET) to measure localized Rac activation associated with IRSp53 and spinophilin complexes in individual fibroblasts to test this hypothesis. Pervanadate or platelet-derived growth factor induced localized Rac activation dependent on Tiam1 and IRSp53. Forskolin or epinephrine induced localized Rac activation dependent on Tiam1 and spinophilin. In spinophilin-deficient cells, Tiam1 co-localized with IRSp53 in response to pervanadate or platelet-derived growth factor. In IRSp53-deficient cells, Tiam1 co-localized with spinophilin in response to forskolin or epinephrine. Total cellular levels of activated Rac were affected only in cells with exogenous Tiam1, and were primarily increased in the membrane fraction. Downstream effects of Rac activation were also stimulus and scaffold-specific. Cell ruffling, spreading, and cell adhesion were dependent on IRSp53, but not spinophilin. Epinephrine decreased IRSp53-dependent adhesion and increased cell migration in a Rac and spinophilin-dependent fashion. These results support the idea that Tiam1 interactions with different scaffold proteins couple distinct upstream signals to localized Rac activation and specific downstream pathways, and suggest that manipulating Tiam1-scaffold interactions can modulate Rac-dependent cellular behaviors.

Microvascular modifications in diabetic retinopathy.

Patients struggling with diabetes are at elevated risks for several sight-threatening diseases, including proliferative diabetic retinopathy (DR). DR manifests in two stages: first, the retinal microvasculature is compromised and capillary degeneration occurs; subsequently, an over-compensatory angiogenic response is initiated. Early changes in the retinal microcirculation include disruptions in blood flow, thickening of basement membrane, eventual loss of mural cells, and the genesis of acellular capillaries. Endothelial apoptosis and capillary dropout lead to a hypoxic inner retina, alterations in growth factors, and upregulation of inflammatory mediators. With disease progression, pathologic angiogenesis generates abnormal preretinal microvessels. Current therapies, which include panretinal photocoagulation and vitrectomy, have remained unaltered for several decades. With several exciting preclinical advances, emergent technologies and innovative cellular targets may offer newfound hope for developing "next-generation" interventional or preventive clinical approaches that will significantly advance current standards of care and clinical outcomes.

Bioactive peptides derived from vascular endothelial cell extracellular matrices promote microvascular morphogenesis and wound healing in vitro.

Studies in our laboratory indicate that collagenase from Clostridium histolyticum promotes endothelial cell and keratinocyte responses to injury in vitro and wound healing in vivo. We postulate that matrix degradation by Clostridial collagenase creates bioactive fragments that can stimulate cellular responses to injury and angiogenesis. To test this hypothesis, we performed limited digestion of defined capillary-endothelial-derived extracellular matrices using purified human or bacterial collagenases. Immunoprecipitation with antibodies recognizing collagens I, II, III, IV, and V, followed by mass spectrometry reveals the presence of unique fragments in bacterial, but not human-enzyme-digested matrix. Results show that there are several bioactive peptides liberated from Clostridial collagenase-treated matrices, which facilitate endothelial responses to injury, and accelerate microvascular remodeling in vitro. Fragments of collagen IV, fibrillin-1, tenascin X, and a novel peptide created by combining specific amino acids contained within fibrillin 1 and tenascin X each have profound proangiogenic properties. The peptides used at 10-100 nM increase rates of microvascular endothelial cell proliferation by up to 47% and in vitro angiogenesis by 200% when compared with serum-stimulated controls. Current studies are aimed at revealing the molecular mechanisms regulating peptide-induced wound healing while extending these in vitro observations using animal modeling.

Dynamic reciprocity in the wound microenvironment.

Here, we define dynamic reciprocity (DR) as an ongoing, bidirectional interaction among cells and their surrounding microenvironment. In this review, we posit that DR is especially meaningful during wound healing as the DR-driven biochemical, biophysical, and cellular responses to injury play pivotal roles in regulating tissue regenerative responses. Such cell-extracellular matrix interactions not only guide and regulate cellular morphology, but also cellular differentiation, migration, proliferation, and survival during tissue development, including, e.g., embryogenesis, angiogenesis, as well as during pathologic processes including cancer, diabetes, hypertension, and chronic wound healing. Herein, we examine DR within the wound microenvironment while considering specific examples across acute and chronic wound healing. This review also considers how a number of hypotheses that attempt to explain chronic wound pathophysiology may be understood within the DR framework. The implications of applying the principles of DR to optimize wound care practice and future development of innovative wound healing therapeutics are also briefly considered.

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.

Blood-Brain Barrier Damage in Ischemic Stroke and Its Regulation by Endothelial Mechanotransduction.

Ischemic stroke, a major cause of mortality in the United States, often contributes to disruption of the blood-brain barrier (BBB). The BBB along with its supportive cells, collectively referred to as the "neurovascular unit," is the brain's multicellular microvasculature that bi-directionally regulates the transport of blood, ions, oxygen, and cells from the circulation into the brain. It is thus vital for the maintenance of central nervous system homeostasis. BBB disruption, which is associated with the altered expression of tight junction proteins and BBB transporters, is believed to exacerbate brain injury caused by ischemic stroke and limits the therapeutic potential of current clinical therapies, such as recombinant tissue plasminogen activator. Accumulating evidence suggests that endothelial mechanobiology, the conversion of mechanical forces into biochemical signals, helps regulate function of the peripheral vasculature and may similarly maintain BBB integrity. For example, the endothelial glycocalyx (GCX), a glycoprotein-proteoglycan layer extending into the lumen of bloods vessel, is abundantly expressed on endothelial cells of the BBB and has been shown to regulate BBB permeability. In this review, we will focus on our understanding of the mechanisms underlying BBB damage after ischemic stroke, highlighting current and potential future novel pharmacological strategies for BBB protection and recovery. Finally, we will address the current knowledge of endothelial mechanotransduction in BBB maintenance, specifically focusing on a potential role of the endothelial GCX.

The pericyte: cellular regulator of microvascular blood flow.

The vascular system - through its development, response to injury, and remodeling during disease - constitutes one of the key organ systems sustaining normal human physiology; conversely, its dysregulation also underlies multiple pathophysiologic processes. Regulation of vascular endothelial cell function requires the integration of complex signals via multiple cell types, including arterial smooth muscle, capillary and post-capillary pericytes, and other perivascular cells such as glial and immune cells. Here, we focus on the pericyte and its roles in microvascular remodeling, reviewing current concepts in microvascular pathophysiology and offering new insights into the specific roles that pericyte-dependent signaling pathways may play in modulating endothelial growth and microvascular tone during pathologic angiogenesis and essential hypertension.

Inhibition of angiogenesis in vitro: a central role for beta-actin dependent cytoskeletal remodeling.

Endothelial cell migration and proliferation, central steps in both physiologic and pathologic angiogenesis, require cytoskeletal-dependent remodeling, which is, in large part, achieved by the dynamic regulation of the beta-actin network. Specifically, the beta-actin network has previously been shown to be (i) enriched in regions of highly motile cytoplasm, and (ii) modulated by its isoactin-specific barbed-end capping protein, beta cap73. We hypothesize that regulated over-expression of beta cap73 could disrupt angiogenesis by capping beta-actin-filament assembly thus inhibiting the incipient cellular migration and microvascular morphogenesis that ensues. Indeed, upon infection of capillary endothelial cells (cEC) with an adenovirus encoding the full-length beta cap73 (Ad-beta cap73), there is a robust cellular rounding response that occurs concomitantly with cytoskeletal disruption, as visualized with immunofluorescence microscopy. Further, we demonstrate that over-expression of Ad-beta cap73 inhibits cEC migration in wound healing studies. Quantitative in vitro angiogenesis assays reveal that Ad-beta cap73 not only prevents endothelial cells from forming capillary-like networks, but also induces the collapse of preformed endothelial tubes. In testing whether Ad-beta cap73 impairs angiogenic events by inducing anoikis/apoptosis, we demonstrate that beta cap73 infection activates a caspase-3-mediated cell death response as observed by quantitative Western blotting and immunofluorescence analyses. Altogether, these findings suggest that endothelial-specific targeting and beta cap73 over-expression may represent an innovative therapeutic approach capable of abrogating pathologic angiogenesis.

Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone.

The myosin family of motor proteins is implicated in mediating actin-based growth cone motility, but the roles of many myosins remain unclear. We previously implicated myosin 1c (M1c; formerly myosin I beta) in the retention of lamellipodia (Wang et al., 1996). Here we address the role of myosin II (MII) in chick dorsal root ganglion neuronal growth cone motility and the contribution of M1c and MII to retrograde F-actin flow using chromophore-assisted laser inactivation (CALI). CALI of MII reduced neurite outgrowth and growth cone area by 25%, suggesting a role for MII in lamellipodial expansion. Micro-CALI of MII caused a rapid reduction in local lamellipodial protrusion in growth cones with no effects on filopodial dynamics. This is opposite to micro-CALI of M1c, which caused an increase in lamellipodial protrusion. We used fiduciary beads (Forscher et al., 1992) to observe retrograde F-actin flow during the acute loss of M1c or MII. Micro-CALI of M1c reduced retrograde bead flow by 76%, whereas micro-CALI of MII or the MIIB isoform did not. Thus, M1c and MIIB serve opposite and nonredundant roles in regulating lamellipodial dynamics, and M1c activity is specifically required for retrograde F-actin flow.

EspFU, a type III-translocated effector of actin assembly, fosters epithelial association and late-stage intestinal colonization by E. coli O157:H7.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 induces filamentous actin-rich 'pedestals' on intestinal epithelial cells. Pedestal formation in vitro requires translocation of bacterial effectors into the host cell, including Tir, an EHEC receptor, and EspF(U), which increases the efficiency of actin assembly initiated by Tir. While inactivation of espF(U) does not alter colonization in two reservoir hosts, we utilized two disease models to explore the significance of EspF(U)-promoted actin pedestal formation. EHECDeltaespF(U) efficiently colonized the rabbit intestine during co-infection with wild-type EHEC, but co-infection studies on cultured cells suggested that EspF(U) produced by wild-type bacteria might have rescued the mutant. Significantly, EHECDeltaespF(U) by itself was fully capable of establishing colonization at 2 days post inoculation but unlike wild type, failed to expand in numbers in the caecum and colon by 7 days. In the gnotobiotic piglet model, an espF(U) deletion mutant appeared to generate actin pedestals with lower efficiency than wild type. Furthermore, aggregates of the mutant occupied a significantly smaller area of the intestinal epithelial surface than those of the wild type. Together, these findings suggest that, after initial EHEC colonization of the intestinal surface, EspF(U) may stabilize bacterial association with the epithelial cytoskeleton and promote expansion beyond initial sites of infection.

Creation of human skin equivalents for the in vitro study of angiogenesis in wound healing.

In our efforts aimed at studying the cellular responses to injury, including the angiogenesis of wound healing, we have developed a novel three-dimensional (3D) skin equivalent that is comprised of multiple cell types found in normal human skin or chronic wound beds. The in vitro model contains a microvascular component within the dermis-like extracellular matrix and possesses an intact epithelial covering comprised of skin-derived epithelial cells. Capillary endothelial cells can be labeled with fluorescent vital tracers prior to being embedded within a 3D matrix and overlaid with a monolayer of keratinocytes (normal or transformed). Once embedded in the matrix, the endothelial cells demonstrate capillary-like tube formation mimicking the microvasculature of true skin. Angiogenesis and the reepithelialization, which occur in response to injury and during wound healing, can be quantified using fluorescence-based and bright-field digital imaging microscopic, biochemical, or molecular approaches.