Anterior Cruciate Ligament Reconstruction using Bone-Tendon-Bone Allograft: Surgical Technique Using Augmentation with Bio-Composite Scaffold.

Anterior cruciate ligament (ACL) rupture rates remain high; the incidence of isolated ACL ruptures is 68.6 per 100,000. This Technical Note introduces a technique for ACL reconstruction (ACLR) using a bone-tendon-bone (BTB) allograft augmented with BioBrace, a biocomposite scaffold. The BioBrace scaffold is sutured onto the BTB allograft to reinforce the ligament and accelerate healing. Graft preparation with BioBrace, ACLR, and graft passage is described. This technique aims to reduce re-rupture risk, enhance graft healing, and improve patient-reported outcomes. BioBrace offers advantages over other augmentation approaches and synthetic materials, providing improved remodeling, biologic integration, and increased mechanical strength. Feasibility and efficacy have been demonstrated in animal models and human applications. This technique presents a promising approach to enhance ACLR outcomes.

Eccentric Hamstring Strength Imbalance among Football and Soccer Athletes.

OBJECTIVES: Hamstring strain injuries (HSI) are common among football and soccer athletes. Eccentric strength imbalance is considered a contributing factor for HSI. There is, however, a paucity of data on hamstring imbalances of soccer and American football athletes as they age and advance in skill level. High school athletes will display greater interlimb discrepancies compared with collegiate and professional athletes. In addition, soccer athletes will exhibit greater hamstring asymmetry than American football athletes. METHODS: Hamstring testing was performed on soccer and American football athletes using the NordBord Hamstring Testing System (Vald Performance, Albion, Australia). Age, sex, weight, sport specialization, and sport level were recorded. Maximum hamstring forces (N), torque (N · m), and work (N · s) were measured. Hamstring imbalance (%) was calculated by dividing the absolute value of the difference in leg forces divided by their sum. One-way analysis of variance and independent sample t tests compared measurements between athlete groups. RESULTS: A total of 631 athletes completed measurements, including 88 high school male soccer, 25 college male soccer, 23 professional male soccer, 83 high school female soccer, 28 college female soccer, 288 high school football, and 96 college football athletes. High school soccer players displayed significantly greater imbalances for torque (P = 0.03) and work (P < 0.01) than football athletes. Imbalances for maximum force (P = 0.035), torque (P = 0.018), and work (P = 0.033) were significantly higher for male soccer athletes in high school compared with college- and professional-level athletes. Female high school soccer players had significantly higher imbalance in torque (P = 0.045) and work (P = 0.001) compared with female collegiate soccer players. Football athletes did not experience significant changes in force imbalances between skill levels. CONCLUSIONS: High school soccer athletes exhibit greater hamstring imbalances than football athletes. Higher levels of play in soccer, for both male and female athletes, correlate with less hamstring asymmetry.

Anchor-Based Meniscal Ramp Repair.

Ramp lesions of the medial meniscus are underdiagnosed because of difficulty in visualizing via magnetic resonance imaging and during arthroscopy. They most often occur simultaneously with anterior cruciate ligament (ACL) injury but may also be associated with posterior plateau contusions, steeper medial tibial plateau slope, and excess varus alignment. Upwards of 24% of ACL reconstructions have concomitant ramp lesions. Failure to repair the ramp lesion is associated with increased rotational laxity, tibial translocation, persistent pivot shift, and poorer outcomes after ACL reconstruction. The purpose of this article is to describe an all-suture anchor-based repair of a meniscal ramp lesion, which confers several advantages over traditional repair techniques.

The Influence of Psychosocial Factors on Patients Undergoing Anterior Cruciate Ligament Reconstruction.

CONTEXT: Anterior cruciate ligament (ACL) injuries greatly impact patients in terms of future performance, reduced physical activity and athletic participation, and overall economic burden. Decades of research have investigated how to improve ACL reconstruction (ACLR) outcomes. Recently, there has been growing interest to understand the effects of psychosocial factors on patient outcomes. STUDY DESIGN: Clinical review. EVIDENCE ACQUISITION: A search of the PubMed database was performed in March 2023. Articles were reviewed by at least 2 authors to determine relevance. We highlighted publications of the past 5 years while incorporating previous pertinent studies. LEVEL OF EVIDENCE: Level 5. RESULTS: There is no standardization of psychosocial factors regarding ACLR. As such, there is a lack of consensus regarding which psychosocial measures to use and when. There is a need for clarification of the complex relationship between psychosocial factors and physical function. Despite this, psychosocial factors have the potential to help predict patients who are more likely to return to sport: (1) desire/motivation to return; (2) lower levels of kinesiophobia; (3) higher levels of self-efficacy, confidence, and subjective knee function; (4) risk acceptance; and (5) social support. However, there are no standardized interventions to improve psychosocial factors after ACLR. CONCLUSION: Psychosocial factors affect outcomes after ACLR. However, the interplay between psychosocial factors and physical function is complex. There is emerging evidence that testing and interventions may improve ACLR outcomes. There is a lack of standardized interventions to determine or improve psychosocial factors after ACLR. Further research is needed to identify psychosocial factors and to develop standardized interventions for clinicians to implement to improve clinical outcomes.

The Impact of Smoking on Hospital Course and Postoperative Outcomes in Patients With Fracture-Related Infections.

OBJECTIVES: To assess the relationship between patient smoking status and fracture-related infection (FRI) characteristics including patient symptoms at FRI presentation, bacterial species of FRI, and rates of fracture union. DESIGN: Retrospective cohort study. SETTING: Urban level 1 trauma center. PATIENT SELECTION CRITERIA: All patients undergoing reoperation for FRI from January 2013 to April 2021 were identified through manual review of an institutional database. OUTCOME MEASURES AND COMPARISONS: Data including patient demographics, fracture characteristics, infection presentation, and hospital course were collected through review of the electronic medical record. Patients were grouped based on current smoker versus nonsmoker status. Hospital course and postoperative outcomes of these groups were then compared. Risk factors of methicillin-resistant Staphylococcus aureus (MRSA) infection, Staphylococcus epidermidis infection, and sinus tract development were evaluated using multivariable logistic regression. RESULTS: A total of 301 patients, comprising 155 smokers (51%) and 146 nonsmokers (49%), undergoing FRI reoperation were included. Compared with nonsmokers, smokers were more likely male (69% vs. 56%, P = 0.024), were younger at the time of FRI reoperation (41.7 vs. 49.5 years, P < 0.001), and had lower mean body mass index (27.2 vs. 32.0, P < 0.001). Smokers also had lower prevalence of diabetes mellitus (13% vs. 25%, P = 0.008) and had higher Charlson Comorbidity Index 10-year estimated survival (93% vs. 81%, P < 0.001). Smokers had a lower proportion of S. epidermidis infections (11% vs. 20%, P = 0.037), higher risk of nonunion after index fracture surgery (74% vs. 61%, P = 0.018), and higher risk of sinus tracts at FRI presentation (38% vs. 23%, P = 0.004). On multivariable analysis, smoking was not found to be associated with increased odds of MRSA infection. CONCLUSIONS: Among patients who develop a FRI, smokers seemed to have better baseline health regarding age, body mass index, diabetes mellitus, and Charlson Comorbidity Index 10-year estimated survival compared with nonsmokers. Smoking status was not significantly associated with odds of MRSA infection. However, smoking status was associated with increased risk of sinus tract development and nonunion and lower rates of S. epidermidis infection at the time of FRI reoperation. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Nuclear SUN1 stabilizes endothelial cell junctions via microtubules to regulate blood vessel formation.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cytoskeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity.

Modeling Occupational Fingernail Onycholysis Disorders in the Population of US Astronauts Who Have Engaged in Extravehicular Activity.

OBJECTIVES: Spacesuits are designed to be reliable personal spacecraft that preserve the life and well-being of the astronaut from the extremes of space. However, materials, operating pressures, and suit design requirements often result in a risk of musculoskeletal discomfort and injury to various areas of the body. In particular, this investigation looked at fingernails and their risk of developing onycholysis. METHODS: An onycholysis literature review was followed by a retrospective analysis of injury characteristics, astronaut suited training and spaceflight events, hand anthropometry, glove sizing, and astronaut demographics. Multiple logistic regression was used to assess the likelihood of onycholysis occurrence by testing potential risk variables against the dataset compiled from the retrospective data mining. RESULTS: The duration of event exposure, type of glove used, distance (delta) between the fingertip and the tip of the glove, sex, and age were found to be significantly related to occurrence of onycholysis (whether protective or injurious). CONCLUSION: An initial risk formula (model) for onycholysis was developed as a result of this investigation. In addition to validation through a future study, further improvement to this onycholysis equation and spacesuit discomfort and injury in general can be aided by future investigations that lead to better definition of the threshold between safe and risky exposure for each type of risk factor. APPLICATION: This work described a potential method that can be used for EVA spacesuit glove onycholysis injury risk analysis for either iterative glove design or between glove comparisons, such as during a product downselect process.

Spacesuit Center of Gravity Assessments for Partial Gravity EVA Simulation in an Underwater Environment.

OBJECTIVE: The objective is to analytically determine the expected CG and build hardware to measure and verify the suited subject's CG for lunar extravehicular activity (EVA) training in an underwater environment. BACKGROUND: For lunar EVAs, it is necessary for astronauts to train with a spacesuit in a simulated partial gravity environment. NASA's Neutral Buoyancy Laboratory (NBL) can provide these conditions by producing negative buoyancy for a submerged suited subject. However, it is critical that the center of gravity (CG) for the human-spacesuit system to be accurate for conditions expected during planetary EVAs. METHODS: An underwater force-transducer system and individualized human-spacesuit model was created to provide real-time measurement of CG, including recommendations for weight placement locations and quantity of weight needed on the spacesuit to achieve a realistic lunar spacesuit CG. This method was tested with four suited subjects. RESULTS: Across tested weighout configurations, it was observed that an aft and high CG location will have large postural differences when compared to low and fore CG locations, highlighting the importance of having a proper CG. The system had an accuracy of ±5lbs of the total lunar weight and within ± 15 cm for fore-aft and left-right CG directions of the model predictions. CONCLUSION: The developed method offers analytical verification of the suited subject's CG and improves simulation quality of lunar EVAs. Future suit design can also benefit by recommending hardware changes to create ideal CG locations that improve balance and mobility. APPLICATION: The developed methodology can be used to verify a proper CG location in future planetary EVA simulations such as different reduced gravity training analogs (e.g. active cable offloading systems).

Novel Use of a Shoulder Spica: Treatment for Midshaft Humerus Fracture in the Presence of Limb Deficiency.

Pediatric midshaft humerus fractures are typically managed with a hanging arm cast, Sarmiento bracing, coaptation splint, or a combination of these treatment options. Here we report a novel use of a shoulder spica cast in the treatment of a midshaft humerus fracture in the presence of limb deficiency. Current treatments proved unsuccessful in maintaining adequate alignment, specifically the varus deformity of the fracture. A shoulder spica was able to successfully maintain acceptable alignment throughout the duration of the patient's healing process. This nontraditional use of a shoulder spica cast shows the practicality of its ability to be utilized for the treatment of unique upper extremity orthopedic obstacles.

Delayed Diagnosis of Pediatric Sternoclavicular Joint Infections and Clavicular Osteomyelitis During the COVID-19 Pandemic: A Report of 3 Cases.

Sternoclavicular joint infections and osteomyelitis of the clavicle are extremely rare infections, especially in the pediatric population. Early signs of these infections are nonspecific and can be mistaken for common upper respiratory infections such as COVID-19 and influenza. Rapid diagnosis and treatment are critical for preventing potentially fatal complications such as mediastinitis. We present three cases of sternoclavicular joint infections in the past year during the COVID-19 pandemic. All three patients had delayed diagnoses likely secondary to COVID-19 workup. Each patient underwent surgical irrigation and débridement. Two of three patients required multiple surgeries and prolonged antibiotic courses. Placement of antibiotic-impregnated calcium sulfate beads into the surgical site cleared the infection in all cases where they were used. All three patients made a full recovery; however, the severity of their situations should not be overlooked. Children presenting to the hospital with chest pain, fever, and shortness of breath should not simply be discharged based on a negative COVID-19 test or other viral assays. A higher index of suspicion for bacterial infections such as clavicular osteomyelitis is important. Close attention must be placed on the physical examination to locate potential areas of concentrated pain, erythema, or swelling to prompt advanced imaging if necessary.

Characterization and Comparison of Convergence Among Cephalotus follicularis Pitcher Plant-Associated Communities With Those of Nepenthes and Sarracenia Found Worldwide.

The Albany pitcher plant, Cephalotus follicularis, has evolved cup-shaped leaves and a carnivorous habit completely independently from other lineages of pitcher plants. It is the only species in the family Cephalotaceae and is restricted to a small region of Western Australia. Here, we used metabarcoding to characterize the bacterial and eukaryotic communities living in C. follicularis pitchers at two different sites. Bacterial and eukaryotic communities were correlated in both richness and composition; however, the factors associated with richness were not the same across bacteria and eukaryotes, with bacterial richness differing with fluid color, and eukaryotic richness differing with the concentration of DNA extracted from the fluid, a measure roughly related to biomass. For turnover in composition, the variation in both bacterial and eukaryotic communities primarily differed with fluid acidity, fluid color, and sampling site. We compared C. follicularis-associated community diversity with that of Australian Nepenthes mirabilis, as well as a global comparison of Southeast Asian Nepenthes and North American Sarracenia. Our results showed similarity in richness with communities from other pitcher plants, and specific bacterial taxa shared among all three independent lineages of pitcher plants. Overall, we saw convergence in richness and particular clades colonizing pitcher plants around the world, suggesting that these highly specialized habitats select for certain numbers and types of inhabitants.

Indigenizing our research: indigenous community leadership in HIV epidemiology research.

The use of data intensive health research has allowed for greater understandings of population health. When conducting data intensive health research, engaging and involving the community is essential for conducting meaningful research that is responsive to the public's needs. Particularly, when engaging Indigenous communities in research, there is a need to understand historical and ongoing impacts of colonialism and recognize the strengths in Indigenous Peoples' knowledges and experiences while supporting Indigenous leadership and self-determination in research. This article describes the approach our research team/organization used to engage and involve Indigenous people living with HIV in three research projects using large, linked datasets and looking at HIV outcomes of Indigenous populations in Canada. The foundation of these projects was simultaneously: 1) supporting Indigenous people living with HIV to be involved as research team members, 2) developing research questions to answer with available datasets, and 3) integrating Indigenous and Western ways of knowing. We have identified important considerations and suggestions for engaging and involving Indigenous communities and individuals in the generation of research ideas and analysis of linked data using community-based participatory research approaches through our work. These include engaging stakeholders at the start of the project and involving them throughout the research process, honouring Indigenous ways of knowing, the land, and local protocols and traditions, prioritizing Indigenous voices, promoting co-learning and building capacity, and focusing on developing longitudinal relationships. We describe keys to success and learnings that emerged. Importantly, the methodology practiced and presented in this manuscript is not a qualitative study design whereby research subjects are surveyed about their experiences or beliefs. Rather, the study approach described herein is about engaging people with living experience to co-lead as researchers. Our approach supported Indigenous people to share research that addresses their research priorities and responds to issues relevant to Indigenous Peoples and communities.

Building Bridges: Describing a Process for Indigenous Engagement in Epidemiology.

BACKGROUND: Processes for epidemiology embedded with Indigenous methodology are needed. Building Bridges was developed to engage Indigenous peoples in epidemiology to address health issues relevant to them. OBJECTIVES: We describe our process for meaningfully engaging Indigenous leaders and peoples living with human immunodeficiency virus (HIV) in epidemiology research. METHODS: As a community-based research (CBR) project, Indigenous methodologies and leadership ensured the quality and relevance of findings. Study phases included 1) advisory board formation, 2) recruitment, 3) research question identification, 4) data analysis from the Canadian HIV Observational Cohort (CANOC) collaboration, 5) data interpretation and contextualization, and 6) knowledge translation and exchange. LESSONS LEARNED: Support and guidance from Indigenous team members, Spiritual Leaders and Elders along with meaningful relationships with allied academic researchers were pivotal. Expertise and lived experiences in Indigenous culture, HIV, epidemiology and services enabled multidirectional learning. CONCLUSIONS: Building Bridges' success hinged on ongoing co-learning and engagement of Indigenous peoples, service providers and researchers.

The role of endothelial MERTK during the inflammatory response in lungs.

As a key homeostasis regulator in mammals, the MERTK receptor tyrosine kinase is crucial for efferocytosis, a process that requires remodeling of the cell membrane and adjacent actin cytoskeleton. Membrane and cytoskeletal reorganization also occur in endothelial cells during inflammation, particularly during neutrophil transendothelial migration (TEM) and during changes in permeability. However, MERTK's function in endothelial cells remains unclear. This study evaluated the contribution of endothelial MERTK to neutrophil TEM and endothelial barrier function. In vitro experiments using primary human pulmonary microvascular endothelial cells found that neutrophil TEM across the endothelial monolayers was enhanced when MERTK expression in endothelial cells was reduced by siRNA knockdown. Examination of endothelial barrier function revealed increased passage of dextran across the MERTK-depleted monolayers, suggesting that MERTK helps maintain endothelial barrier function. MERTK knockdown also altered adherens junction structure, decreased junction protein levels, and reduced basal Rac1 activity in endothelial cells, providing potential mechanisms of how MERTK regulates endothelial barrier function. To study MERTK's function in vivo, inflammation in the lungs of global Mertk-/- mice was examined during acute pneumonia. In response to P. aeruginosa, more neutrophils were recruited to the lungs of Mertk-/- than wildtype mice. Vascular leakage of Evans blue dye into the lung tissue was also greater in Mertk-/- mice. To analyze endothelial MERTK's involvement in these processes, we generated inducible endothelial cell-specific (iEC) Mertk-/- mice. When similarly challenged with P. aeruginosa, iEC Mertk-/- mice demonstrated no difference in neutrophil TEM into the inflamed lungs or in vascular permeability compared to control mice. These results suggest that deletion of MERTK in human pulmonary microvascular endothelial cells in vitro and in all cells in vivo aggravates the inflammatory response. However, selective MERTK deletion in endothelial cells in vivo failed to replicate this response.

Mechanotransduction: from the cell surface to the nucleus via RhoA.

Cells respond and adapt to their physical environments and to the mechanical forces that they experience. The translation of physical forces into biochemical signalling pathways is known as mechanotransduction. In this review, we focus on two aspects of mechanotransduction. First, we consider how forces exerted on cell adhesion molecules at the cell surface regulate the RhoA signalling pathway by controlling the activities of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In the second part of the review, we discuss how the nucleus contributes to mechanotransduction as a physical structure connected to the cytoskeleton. We focus on recent studies that have either severed the connections between the nucleus and the cytoskeleton, or that have entirely removed the nucleus from cells. These actions reduce the levels of active RhoA, thereby altering the mechanical properties of cells and decreasing their ability to generate tension and respond to external mechanical forces. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

A Rnd3/p190RhoGAP pathway regulates RhoA activity in idiopathic pulmonary fibrosis fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is an incurable disease of the lung that is characterized by excessive deposition of extracellular matrix (ECM), resulting in disruption of normal lung function. The signals regulating fibrosis include both transforming growth factor beta (TGF-ß) and tissue rigidity and a major signaling pathway implicated in fibrosis involves activation of the GTPase RhoA. During studies exploring how elevated RhoA activity is sustained in IPF, we discovered that not only is RhoA activated by profibrotic stimuli but also that the expression of Rnd3, a major antagonist of RhoA activity, and the activity of p190RhoGAP (p190), a Rnd3 effector, are both suppressed in IPF fibroblasts. Restoration of Rnd3 levels in IPF fibroblasts results in an increase in p190 activity, a decrease in RhoA activity and a decrease in the overall fibrotic phenotype. We also find that treatment with IPF drugs nintedanib and pirfenidone decreases the fibrotic phenotype and RhoA activity through up-regulation of Rnd3 expression and p190 activity. These data provide evidence for a pathway in IPF where fibroblasts down-regulate Rnd3 levels and p190 activity to enhance RhoA activity and drive the fibrotic phenotype.

Increased mortality among Indigenous persons in a multisite cohort of people living with HIV in Canada.

OBJECTIVE: Compare all-cause mortality between Indigenous participants and participants of other ethnicities living with HIV initiating combination antiretroviral therapy (cART) in an interprovincial multi-site cohort. METHODS: The Canadian Observational Cohort is a collaboration of 8 cohorts of treatment-naïve persons with HIV initiating cART after January 1, 2000. Participants were followed from the cART initiation date until death or last viral load (VL) test date on or before December 31, 2012. Cox proportional hazard models were used to estimate the effect of ethnicity on time until death after adjusting for age, gender, injection drug use, being a man who has sex with men, hepatitis C, province of origin, baseline VL and CD4 count, year of cART initiation and class of antiretroviral medication. RESULTS: The study sample consisted of 7080 participants (497 Indigenous, 2471 Caucasian, 787 African/Caribbean/Black (ACB), 629 other, and 2696 unknown ethnicity). Most Indigenous persons were from British Columbia (BC) (83%), with smaller numbers from Ontario (13%) and Québec (4%). During the study period, 714 (10%) participants died. The five-year survival probability was lower for Indigenous persons (0.77) than for Caucasian (0.94), ACB (0.98), other ethnicities (0.96) and unknown ethnicities (0.85) (p < 0.0001). In an adjusted proportional hazard model for which missing data were imputed, Indigenous persons were more likely to die than Caucasian participants (hazard ratio = 2.69, p < 0.0001). CONCLUSION: The mortality rate for Indigenous persons was higher than for other ethnicities and is largely reflective of the BC population. Addressing treatment challenges and identifying HIV- and non-HIV-related causes for mortality among Indigenous persons is required to optimize their clinical management.

Tumor Endothelial Cells with Distinct Patterns of TGFß-Driven Endothelial-to-Mesenchymal Transition.

Endothelial-to-mesenchymal transition (EndMT) occurs during development and underlies the pathophysiology of multiple diseases. In tumors, unscheduled EndMT generates cancer-associated myofibroblasts that fuel inflammation and fibrosis, and may contribute to vascular dysfunction that promotes tumor progression. We report that freshly isolated subpopulations of tumor-specific endothelial cells (TEC) from a spontaneous mammary tumor model undergo distinct forms of EndMT in response to TGFß stimulation. Although some TECs strikingly upregulate α smooth muscle actin (SMA), a principal marker of EndMT and activated myofibroblasts, counterpart normal mammary gland endothelial cells (NEC) showed little change in SMA expression after TGFß treatment. Compared with NECs, SMA(+) TECs were 40% less motile in wound-healing assays and formed more stable vascular-like networks in vitro when challenged with TGFß. Lineage tracing using ZsGreen(Cdh5-Cre) reporter mice confirmed that only a fraction of vessels in breast tumors contain SMA(+) TECs, suggesting that not all endothelial cells (EC) respond identically to TGFß in vivo. Indeed, examination of 84 TGFß-regulated target genes revealed entirely different genetic signatures in TGFß-stimulated NEC and TEC cultures. Finally, we found that basic FGF (bFGF) exerts potent inhibitory effects on many TGFß-regulated genes but operates in tandem with TGFß to upregulate others. ECs challenged with TGFß secrete bFGF, which blocks SMA expression in secondary cultures, suggesting a cell-autonomous or lateral-inhibitory mechanism for impeding mesenchymal differentiation. Together, our results suggest that TGFß-driven EndMT produces a spectrum of EC phenotypes with different functions that could underlie the plasticity and heterogeneity of the tumor vasculature.

The RhoA guanine nucleotide exchange factor, LARG, mediates ICAM-1-dependent mechanotransduction in endothelial cells to stimulate transendothelial migration.

RhoA-mediated cytoskeletal rearrangements in endothelial cells (ECs) play an active role in leukocyte transendothelial cell migration (TEM), a normal physiological process in which leukocytes cross the endothelium to enter the underlying tissue. Although much has been learned about RhoA signaling pathways downstream from ICAM-1 in ECs, little is known about the consequences of the tractional forces that leukocytes generate on ECs as they migrate over the surface before TEM. We have found that after applying mechanical forces to ICAM-1 clusters, there is an increase in cellular stiffening and enhanced RhoA signaling compared with ICAM-1 clustering alone. We have identified that leukemia-associated Rho guanine nucleotide exchange factor (LARG), also known as Rho GEF 12 (ARHGEF12) acts downstream of clustered ICAM-1 to increase RhoA activity, and that this pathway is further enhanced by mechanical force on ICAM-1. Depletion of LARG decreases leukocyte crawling and inhibits TEM. To our knowledge, this is the first report of endothelial LARG regulating leukocyte behavior and EC stiffening in response to tractional forces generated by leukocytes.

VE-cadherin status as an indicator of microvascular permeability.

VE-cadherin phosphorylation and binding partner status are important indicators of endothelial permeability. Here we describe several techniques aimed at discerning total tyrosine phosphorylation levels of VE-cadherin, VE-cadherin phosphorylation on specific tyrosine residues, and the ability of VE-cadherin to bind its binding partner beta-catenin. Taken together, these approaches to studying VE-cadherin status on microvascular endothelial cells are excellent complements to traditional permeability assays.