Collaborative Enhancement of Antibody Binding to Distinct PECAM-1 Epitopes Modulates Endothelial Targeting

Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitate targeted drug delivery to endothelial cells by “vascular immunotargeting.” To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs) to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The “collaborative enhancement” of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The “collaborative enhancement” phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents

Visual art instruction in medical education: a narrative review

The humanities have been increasingly incorporated into medical school curricula in order to promote clinical skills and professional formation. To understand its current use, we reviewed the literature on visual arts training in medical education, including relevant qualitative and quantitative data. Common themes that emerged from this review included a focus on preclinical students; instruction promoting observation, diagnostic skills, empathy, team building, communication skills, resilience, and cultural sensitivity. Successful partnerships have involved local art museums, with sessions led primarily by art educators employing validated pedagogy such as Visual Thinking Strategies or Artful Thinking. There is evidence that structured visual arts curricula can facilitate the development of clinical observational skills, although these studies are limited in that they have been single-institution reports, short term, involved small numbers of students and often lacked controls. There is a paucity of rigorous published data demonstrating that medial student art education training promotes empathy, team building, communication skills, wellness and resilience, or cultural sensitivity. Given these concerns, recommendations are offered for fostering more robust, evidence-based approaches for using visual arts instruction in the training of medical students

SHP-2 phosphatase activity is required for PECAM-1-dependent cell motility

Platelet endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in endothelial cell motility during angiogenesis. Although there is evidence that SHP-2 plays a role in PECAM-1-dependent cell motility, the molecular basis of the activity of SHP-2 in this process has not been defined. To investigate the requirement of SHP-2 in PECAM-1-dependent cell motility, studies were done in which various constructs of SHP-2 were expressed in cell transfectants expressing PECAM-1. We observed that the levels of PECAM-1 tyrosine phosphorylation and SHP-2 association with PECAM-1 were significantly increased in cells expressing a phosphatase-inactive SHP-2 mutant, suggesting that the level of PECAM-1 tyrosine phosphorylation, and thus SHP-2 binding are regulated in part by bound, catalytically active SHP-2. We subsequently found that expression of PECAM-1 stimulated wound-induced migration and the formation of filopodia (a morphological feature of motile cells). These activities were associated with increased mitogen-activated protein kinase (MAPK) activation and the dephosphorylation of paxillin (an event implicated in the activation of MAPK). The phosphatase-inactive SHP-2 mutant, however, suppressed these PECAM-1-dependent phenomena, whereas the activity of PECAM-1 expressing cells was not altered by expression of wild-type SHP-2 or SHP-2 in which the scaffold/adaptor function had been disabled. Pharmacological inhibition of SHP-2 phosphatase activity also suppressed PECAM-1-dependent motility. Furthermore, PECAM-1 expression also stimulates tube formation, but none of the SHP-2 constructs affected this process. These findings therefore suggest a model for the involvement of SHP-2 in PECAM-1-dependent motility in which SHP-2, recruited by its interaction with PECAM-1, targets paxillin to ultimately activate the MAPK pathway and downstream events required for cell motility

Isolation of murine lung endothelial cells

Several protocols for the isolation of endothelial cells (ECs) from murine lung have been described in the literature. We, however, encountered a number of problems while using these procedures that prevented us from consistently or reliably obtaining pure populations of ECs from the lungs of mice. By incorporating specific elements from previously published protocols, as well as adding some novel features, we developed a new strategy for isolating ECs from murine lung. In this approach, a suspension of lung cells is initially prepared from the lungs of 7- to 14-day-old mouse pups using procedures that prevent intravascular clotting and leukocyte activation, minimize mechanical trauma to the lung tissue, and limit exposure to the digesting enzymes. The resulting cell suspension is cultured for 2–3 days, trypsinized to produce a suspension of single cells, and then subjected to fluorescence-activated cell sorting using an anti-ICAM-2 antibody. The sorted cells are then plated and split 1:2 at each passage to maintain a high density of the cells. Using this approach, we have been able to isolate pure populations of ECs that were sustainable for extended periods in culture without the emergence of fibroblast overgrowth or the development of senescence. We believe the success of this approach will provide opportunities to take advantage of the large and growing number of knockout and transgenic mouse lines to investigate the endothelial-specific roles of targeted molecules in the pulmonary vasculature

Food insecurity and hypertension: A systematic review and meta-analysis

Background: Food insecurity (FIS) is an important public health issue associated with cardiovascular risk. Given the association of FIS with diets of poorer nutritional quality and higher salt intake as well as chronic stress, numerous studies have explored the link between FIS and hypertension. However, no systematic review or meta-analysis has yet to integrate or analyze the existing literature. Methods: We performed a wide and inclusive search of peer-reviewed quantitative data exploring FIS and hypertension. A broad-terms, systematic search of the literature was conducted in PubMed, Embase, Scopus, and Web of Science for all English-language, human studies containing primary data on the relationship between FIS and hypertension. Patient population characteristics, study size, and method to explore hypertension were extracted from each study. Effect sizes including odds ratios and standardized mean differences were extracted or calculated based on studies’ primary data. Comparable studies were combined by the random effects model for meta-analyses along with assessment of heterogeneity and publication bias. Results: A total of 36 studies were included in the final analyses. The studies were combined into different subgroups for meta-analyses as there were important differences in patient population characteristics, methodology to assess hypertension, and choice of effect size reporting (or calculability from primary data). For adults, there were no significantly increased odds of elevated blood pressures for food insecure individuals in studies where researchers measured the blood pressures: OR = 0.91 [95%CI: 0.79, 1.04; n = 29,781; Q(df = 6) = 7.6; I2 = 21%]. This remained true upon analysis of studies which adjusted for subject BMI. Similarly, in studies for which the standardized mean difference was calculable, there was no significant difference in measured blood pressures between food secure and FIS individuals: g = 0.00 [95%CI: -0.04, 0.05; n = 12,122; Q(df = 4) = 3.6; I2 = 0%]. As for retrospective studies that inspected medical records for diagnosis of hypertension, there were no significantly increased odds of hypertension in food insecure adults: OR = 1.11 [95%CI: 0.86, 1.42; n = 2,887; Q(df = 2) = 0.7; I2 = 0%]. In contrast, there was a significant association between food insecurity and self-reports of previous diagnoses of hypertension: 1.46 [95%CI: 1.13, 1.88; n = 127,467; Q(df = 7) = 235; I2 = 97%]. Only five pediatric studies were identified which together showed a significant association between FIS and hypertension: OR = 1.44 [95%CI: 1.16, 1.79; n = 19,038; Q(df = 4) = 5.7; I2 = 30%]. However, the small number of pediatric studies were not sufficient for subgroup meta-analyses based on individual study methodologies. Discussion: In this systematic review and meta-analysis, an association was found between adult FIS and self-reported hypertension, but not with hypertension determined by blood pressure measurement or chart review. Further, while there is evidence of an association between FIS and hypertension among pediatric subjects, the limited number of studies precluded a deeper analysis of this association. These data highlight the need for more rigorous and longitudinal investigations of the relationship between FIS and hypertension in adult and pediatric populations.</p

Expression and Role of the Hyaluronan Receptor RHAMM in Inflammation after Bleomycin Injury

Lung injury is associated with increased concentrations of hyaluronan (hyaluronic acid, HA). HA modifies cell behavior through interaction with cell-associated receptors such as receptor for HA-mediated motility (RHAMM, CD168). Using a function blocking anti-RHAMM antibody (R36), we investigated the expression and role of RHAMM in the inflammatory response to intratracheal bleomycin in rats. Immunostaining showed increased expression of RHAMM in macrophages 4–7 d after injury. Surface biotin labeling of cells isolated by lavage confirmed increased surface expression of a 70-kD RHAMM after lung injury, and in situ hybridization demonstrated increased RHAMM mRNA in macrophages responding to injury. Time-lapse cinemicrography demonstrated a 5-fold increase in motility of alveolar macrophages from bleomycin-treated animals that was completely blocked by R36 in vitro. Further, HA-stimulated macrophage chemotaxis was also inhibited by R36. Daily administration of R36 to injured animals resulted in a 40% decrease in macrophage accumulation 7 d after injury. Further, H&E staining of tissue sections showed that bleomycin-mediated changes in lung architecture were improved with R36 treatment. Taken together with previous results showing the inhibitory effects of HA-binding peptide on inflammation and fibrosis, we conclude that the interaction of RHAMM with HA is a critical component of the recruitment of inflammatory cells to the lung after injury