Publications

Sigma receptor agonists are suspected to modulate blood pressure in humans. We investigated how modulation of sigma receptors impacts phenylephrine (PE)-induced contraction in human mesenteric arterial rings obtained from human organ donors. This study also explored the relationship between sigma receptor activation, PE-induced arterial contraction, and the history of the organ donor's alcohol use. The concentration responsiveness of PE-induced arterial contraction was tested using wire myography in the absence and presence of the sigma receptor agonist PRE-084, and the sigma receptor antagonists BD-1047 and SM-21. Sigma receptor-1 expression in the arteries was also investigated using an automated capillary electrophoresis system. The results show that PRE-084 elicited a downward shift in the PE concentration-response curve. Notably, this trend only occurred in arteries from donors with histories of non-/light drinking or moderate drinking (P<0.05), but not with arteries obtained from donors with histories of heavy or binge drinking. The sigma receptor-1 antagonist BD-1047 elicited an upward shift in the PE concentration-response curve in arteries from non-/light and moderate drinkers, but not from heavy drinkers. Interestingly, the sigma receptor-2 antagonist caused an upward shift in the PE concentration-response curve in arteries from all three groups of donors. Notably, sigma receptor-1 protein levels were decreased in arteries from heavy drinkers compared to the other groups. Collectively, the findings suggest that sigma receptors in human arteries may promote relaxation. However, heavy alcohol consumption reduces arterial sigma receptor-1 expression and impairs its ability to modulate contraction.

OBJECTIVE: Microvascular hyperpermeability is a serious complication that occurs from hemorrhagic shock and resuscitation (HSR), especially when combined with acute alcohol intoxication (AAI). We tested the hypothesis that administration of Apolipoprotein M (ApoM), a lipocalin that normally resides in plasma high-density lipoproteins (HDL) and a carrier of sphingosine-1-phosphate (S1P), reduces combined AAI and HSR-induced microvascular leakage.

METHODS: An established rat model of AAI/HSR was combined with intravital microscopy to study whether the administration of ApoM in resuscitative fluids reduces microvascular leakage of FITC-albumin. The impact of ApoM on human umbilical vein endothelial cell (HUVEC) monolayer barrier function and junctional integrity was tested, using trans-endothelial electrical resistance (TER) and immunofluorescence labeling of junctional VE-Cadherin, respectively. Immunoprecipitation of ApoM in HUVEC and mass spectrometry of complexes were used to determine potential binding partners. The Rac1 G-LISA assay was used to determine if ApoM causes Rac1 activation in HUVEC.

RESULTS: Compared to sham controls, combined AAI and HSR significantly increased microvascular leakage. Administration of S1P, ApoM, or their combination during resuscitation significantly decreased microvascular leakage. In HUVEC monolayers, with or without alcohol pretreatment, S1P, ApoM, and S1P + ApoM all significantly increased barrier function and improved the junctional integrity of VE-cadherin compromised by alcohol. The small GTPase Rac1 was found to bind with ApoM in HUVEC and was significantly activated within 5 min of ApoM addition.

CONCLUSIONS: The findings suggest that fluid resuscitation with ApoM ameliorates AAI/HSR-induced microvascular leakage. The mechanism involves stabilizing VE-Cadherin junction integrity, which could be caused by Rac1 activation.

The microvascular endothelium has a critical role in regulating the delivery of oxygen, nutrients, and water to the surrounding tissues. Under inflammatory conditions that accompany acute injury or disease, microvascular permeability becomes elevated. When microvascular hyperpermeability becomes uncontrolled or chronic, the excessive escape of plasma proteins into the surrounding tissue disrupts homeostasis and ultimately leads to organ dysfunction. Much remains to be learned about the mechanisms that control microvascular permeability. In addition to in vivo and isolated microvessel methods, the cultured endothelial cell monolayer protocol is an important tool that allows for understanding the specific, endothelial subcellular mechanisms that determine permeability of the endothelium to plasma proteins. In this chapter, two variations of the popular Transwell culture methodology to determine permeability to using fluorescently labeled tracers are presented. The strengths and weaknesses of this approach are also discussed.

Endothelial bioenergetics have emerged as a key regulator of endothelial barrier function. Glycolytic parameters have been linked to barrier enhancement, and interruption with mitochondrial complexes was shown to disrupt endothelial barrier. Therefore, a new technology that has been introduced to assess bioenergetics and metabolism has also made it possible to determine roles of specific energy production pathways in endothelial health. The Seahorse extracellular flux analysis by Agilent technologies is a state of the art tool that has been more frequently used to evaluate bioenergetics of endothelial cells. This chapter includes details about different assays that can be used to study endothelial cells using the Seahorse analyzer and how interpretation of the results can provide novel insight about endothelial metabolism.

As a primary interface between the blood and underlying vascular wall, the endothelial glycocalyx layer is common to all blood vessels and covers the luminal surface of all endothelial cells. The endothelial glycocalyx has important roles as a regulator of microvascular endothelial functions such as mechanotransduction, leukocyte adhesion, and microvascular permeability. Disruption of the molecular structure of the endothelial glycocalyx disturbs physiological, and hemodynamic processes associated with the microvascular wall leads to microvascular hyperpermeability. Studying the glycocalyx is challenging because cultured cells present aberrant glycocalyx structure and tissue fixation techniques lead to the degradation and loss of this fine and delicate layer. Therefore, studying the glycocalyx requires in vivo imaging of the microcirculation. Here we describe two techniques for direct imaging and assessment of the glycocalyx surface layer integrity using intravital microscopy (IVM), a method widely used in the study of the dynamic changes that occur in the microcirculation during inflammation or injury.

The ability to view and record the movements of subcellular structures is a powerful tool that has accelerated the discovery and understanding of signaling mechanisms that control microvascular functions such as the control of endothelial permeability. Advances in molecular biology over the past few decades have facilitated the generation of fusion proteins in which fluorescent reporters based upon the structure of green fluorescent protein can be linked to proteins found in human endothelial cells, such as VE-cadherin or β-actin. These fusion proteins have been found to incorporate into structures alongside their native protein counterparts, allowing the dynamic visualization of how these subcellular structures are modified when cells are challenged with stimuli such as inflammatory mediators. The result of such studies has been a much more advanced view of the complex mechanisms by which endothelial cells maintain barrier properties than previously obtained by only viewing fixed cells labeled by immunofluorescence. Here, we describe our protocols that we have used to view the dynamics of actin filaments using time-lapse microscopy to record endothelial cells expressing GFP-actin and the analysis tools available to quantify dynamics of subcellular structures.

Lymphatic vessels have an active role in draining excess interstitial fluid from organs and serving as conduits for immune cell trafficking to lymph nodes. In the central circulation, the force needed to propel blood forward is generated by the heart. In contrast, lymphatic vessels rely on intrinsic vessel contractions in combination with extrinsic forces for lymph propulsion. The intrinsic pumping features phasic contractions generated by lymphatic smooth muscle. Periodic, bicuspid valves composed of endothelial cells prevent backflow of lymph. This work provides a brief overview of lymph transport, including initial lymph formation along with cellular and molecular mechanisms controlling lymphatic vessel pumping.