Lauren A. Biwer

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function.

Endothelial nitric oxide synthase in the microcirculation

Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)—a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition

In resistance arteries, endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). Endothelial nitric oxide synthase (eNOS) is present in the luminal side of the EC (apical EC) and the basal side of the EC (MEJ). To test if these eNOS pools acted in sync or separately, we co-cultured ECs and SMCs, then stimulated SMCs with phenylephrine (PE). Adrenergic activation causes inositol [1,4,5] triphosphate (IP3) to move from SMC to EC through gap junctions at the MEJ. PE increases MEJ eNOS phosphorylation (eNOS-P) at S1177, but not in EC. Conversely, we used bradykinin (BK) to increase EC calcium; this increased EC eNOS-P but did not affect MEJ eNOS-P. Inhibiting gap junctions abrogated the MEJ eNOS-P after PE, but had no effect on BK eNOS-P. Differential lipid composition between apical EC and MEJ may account for the compartmentalized eNOS-P response. Indeed, DAG and phosphatidylserine are both enriched in MEJ. These lipids are cofactors for PKC activity, which was significantly increased at the MEJ after PE. Because PKC activity also relies on endoplasmic reticulum (ER) calcium release, we used thapsigargin and xestospongin C, BAPTA, and PKC inhibitors, which caused significant decreases in MEJ eNOS-P after PE. Functionally, BK inhibited leukocyte adhesion and PE caused an increase in SMC cGMP. We hypothesize that local lipid composition of the MEJ primes PKC and eNOS-P for stimulation by PE, allowing for compartmentalized function of eNOS in the blood vessel wall.

Endoplasmic reticulum-mediated signalling in cellular microdomains

The endoplasmic reticulum (ER) is a prime mediator of cellular signalling due to its functions as an internal cellular store for calcium, as well as a site for synthesis of proteins and lipids. Its peripheral network of sheets and tubules facilitates calcium and lipid signalling, especially in areas of the cell that are more distant to the main cytoplasmic network. Specific membrane proteins shape the peripheral ER architecture and influence the network stability to project into restricted spaces. The signalling microdomains are anatomically separate from the cytoplasm as a whole and exhibit localized protein, ion channel and cytoskeletal element expression. Signalling can also occur between the ER and other organelles, such as the Golgi or mitochondria. Lipids made in the ER membrane can be sent to the Golgi via specialized transfer proteins and specific phospholipid synthases are enriched at ER–mitochondria junctions to more efficiently expedite phospholipid transfer. As a hub for protein and lipid synthesis, a store for intracellular calcium [Ca2+]i and a mediator of cellular stress, the ER is an important cellular organelle. Its ability to organize into tubules and project into restricted spaces allows for discrete and temporal signalling, which is important for cellular physiology and organism homoeostasis.

Endothelial cell α-globin and its molecular chaperone α-hemoglobin–stabilizing protein regulate arteriolar contractility

Arteriolar endothelial cell–expressed (EC-expressed) &agr;-globin binds endothelial NOS (eNOS) and degrades its enzymatic product, NO, via dioxygenation, thereby lessening the vasodilatory effects of NO on nearby vascular smooth muscle. Although this reaction potentially affects vascular physiology, the mechanisms that regulate &agr;-globin expression and dioxygenase activity in ECs are unknown. Without &bgr;-globin, &agr;-globin is unstable and cytotoxic, particularly in its oxidized form, which is generated by dioxygenation and recycled via endogenous reductases. We show that the molecular chaperone &agr;-hemoglobin–stabilizing protein (AHSP) promotes arteriolar &agr;-globin expression in vivo and facilitates its reduction by eNOS. In Ahsp−/− mice, EC &agr;-globin was decreased by 70%. Ahsp−/− and Hba1−/− mice exhibited similar evidence of increased vascular NO signaling, including arteriolar dilation, blunted &agr;1-adrenergic vasoconstriction, and reduced blood pressure. Purified &agr;-globin bound eNOS or AHSP, but not both together. In ECs in culture, eNOS or AHSP enhanced &agr;-globin expression posttranscriptionally. However, only AHSP prevented oxidized &agr;-globin precipitation in solution. Finally, eNOS reduced AHSP-bound &agr;-globin approximately 6-fold faster than did the major erythrocyte hemoglobin reductases (cytochrome B5 reductase plus cytochrome B5). Our data support a model whereby redox-sensitive shuttling of EC &agr;-globin between AHSP and eNOS regulates EC NO degradation and vascular tone.

Non–Endoplasmic Reticulum–Based Calr (Calreticulin) Can Coordinate Heterocellular Calcium Signaling and Vascular Function

Objective— In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function. Approach and Results— We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr &Dgr;/&Dgr;). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr &Dgr;/&Dgr; mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr &Dgr;/&Dgr; had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER. Conclusions— Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure.

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 µm2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 µm pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.