Making Connexons in the Neurovascular Unit

Less than a decade ago, the term blood–brain barrier underwent a major conceptual change when it was recognized that the cerebrovasculature was anything, but passive in its structure and function. Instead of a layer of bricks separated by heavy mortar, the brain endothelial cell became one of a complement of cell types performing together as a dynamic, integrated unit and functioning as a major contributor to brain homeostasis. Today, this assembly is known as the neurovascular unit (NVU) and consists of endothelial cells, pericytes, astroctyes, and neurons with critical roles for microglia, circulating immune cells and smooth muscle cells as well (Abbott et al, 2010; Neuwelt et al, 2011). As a unit, these cells form a permeability barrier; regulate blood flow, neuronal development, and immune function; and conduct surveillance of neural signaling and cellular activity. The NVU is now implicated in many neurologic disease conditions because a damaged or dysfunctional NVU will lead to dysfunction of the surrounding brain cells and their integrated neurologic functions (Neuwelt et al, 2011; Zlokovic, 2011). Thus, the NVU may be either a cause of neurologic disease or a major, therapy requiring, complicating condition associated with a primary disease.

Few empirical studies directly show many functions and communications between NVU cells, but accumulated inferential evidence is overwhelming. Recent technical achievements in dynamic in-vivo microscopy and advances in cellular and molecular approaches have been critically important for establishing functional and signaling relationships within the NVU. Genetic studies, however, have been infrequently used in the field. A notable exception is the work of Ezan et al (2012) reported in this issue of the Journal of Cerebral Blood Flow and Metabolism. Well-known anatomical studies indicated that astrocytic processes called endfeet virtually cover the entire microvascular wall consisting of endothelial cells and pericytes (Mathiisen et al, 2010). Furthermore, recent studies showed that the perivascular astrocytic endfeet are connected by gap junctions composed of the proteins connexin 30 (Cx30) and connexin 43 (Cx43) (Rouach et al, 2008). These proteins become embedded in the plasma membrane and assemble into hexameric structures that when aligned on adjacent cells form a pore capable of intercellular transfer of ion currents, nutrients and signaling molecules up to several hundred Daltons in molecular weight. By creating a double knockout, Ezan et al were able to investigate for the first time, the relationship between these gap junctions and their role in the NVU. They observed that Cx30 and Cx43 expression occurs postnatally in the endfeet of wild-type animals and is completed before weaning, thus correlating with NVU development. In animals with genetic ablation of Cx30 and Cx43, the endfeet became enlarged, devoid of subcellular structures and reduced in aquaporin-4, an endfeet associated water channel, and β-dystroglycan, a basal lamina associating membrane protein (del Zoppo and Milner, 2006; Nielsen et al, 1997; Wolburg et al, 2009). Interestingly, endothelial cells were structurally normal in appearance including, most notably, their tight junctions. Microvascular permeability to sucrose was also similar to wild-type permeabilities and normal. However, when the Cx30/Cx43 null brains were subjected to elevated hydrostatic pressure and sheer stress, there was a very significant increase in sucrose permeability and extravasation of horseradish peroxidase added to the perfusate. This surprising result indicates that the connexons are important for maintaining endothelial cell tight junction integrity under stress conditions.

These findings have several important features. First, it shows the usefulness of genetic models for studies of NVU structure and function. Such tools are underutilized presently and represent a fertile area for future development. Second, the findings reinforce the notion that the NVU consists of cells that are interdependent and may interact and influence other cells or structures in unexpected ways. It raises the possibilities that gap junction signaling may occur between other NVU cells, including astrocytes and pericytes or pericytes and endothelial cells and so forth. Third, pericellular permeability may be physiologically regulated by yet unknown mechanisms. Finally, the communications via gap junctions raise many questions about the nature of the signaling molecules involved such as Ca²⁺, cyclic nucleotides, prostaglandins, and other cytoplasmic constituents. Clearly, as for any good experiment, the interpretations give rise to many new questions and more topics to explore.