Mono-culture of cells is the predominant form of cell culture for toxicological testing; however, mono-culture results are not always concordant with results obtained in vivo
[22–24]. Co-culture is a promising alternative to mono-culture and provides a more in vivo-like environment for the determination of toxicological results [16, 28–30, 32, 34]. Cross-talk between different cell types in co-culture may elicit cellular reactions that are different from those which occur in mono-culture. Co-culture of SAEC and HMVEC models the alveolar-capillary interaction, which is a critical barrier against inhaled particles such as MWCNT. As MWCNT have been shown to enter the lung epithelial cells in vivo
, the aim of this study was to determine if the interaction of lung epithelial cells with MWCNT in vitro could affect the underlying microvascular. This aim was examined through the use of a relevant alveolar-capillary co-culture model so as to allow cell-cell communication between the epithelial and endothelial cells to analyze the microvasvular effects in a manner more akin to in vivo signaling.
MWCNT were shown previously to enter the lung epithelium in vivo and also be taken up by SAEC in vitro
[13, 14, 41]. In vivo exposure to MWCNT resulted in the majority of MWCNT depositing in the alveolar region following both aspiration and inhalation in vivo exposure, with penetration of MWCNT across the lung epithelium into the alveolar interstitium, and progressive development of fibrosis [13, 14]. Exposure of SAEC to MWCNT resulted in an increase in ROS production, protein phosphorylation, cellular migration, and the release of inflammatory cytokines . Co-culture of SAEC with HMVEC did not appear to alter the ability of SAEC to engulf MWCNT. As SAEC were previously shown to elicit a number of cellular effects upon MWCNT exposure , this study determined that cellular mediators secreted by SAEC could affect surrounding cells. No MWCNT were detected in the HMVEC preparations; therefore, it was determined that direct MWCNT exposure was not the primary source of cellular changes in HMVEC.
ROS, the collective term for the intermediates formed during oxidative metabolism that includes O2
-, hydrogen peroxide (H2O2), hydroxyl radical (·OH), and peroxynitrite (ONO2
-), function as both antimicrobial and key signaling molecules  and are involved in various signaling pathways. Aberrant ROS production can result in indiscriminate damage to DNA, proteins, and lipids in cells . Particularly in the pulmonary system, an overproduction of ROS is correlated with endothelial dysfunction and pulmonary hypertension . The increase of O2
- production in HMVEC indicates that an alteration in ROS signaling in HMVEC may be seen after SAEC exposure to MWCNT.
HMVEC displayed membrane ruffling, actin reorganization, and gap formation between the endothelial cells of the monolayer with increasing time of SAEC exposure to MWCNT. This phenomenon had been previously shown to occur in HMVEC following the direct exposure of endothelial cells to MWCNT and resulted in an increase in vascular permeability . Preservation of pulmonary endothelial monolayer integrity is necessary for efficient pulmonary function, and disruption may allow for the transport of fluid and molecular activators into the alveolar region, thus resulting in decreased gas exchange and inflammation . In vivo exposure of rats suggested endothelial dysfuction following inhalation of MWCNT . Integral to the integrity of the endothelial monolayer is the cytoskeleton of the endothelial cells. The maintenance of proper vascular tone is essential to prevent vascular failure . Loss of actin organization in HMVEC suggested dysfunction in the endothelial monolayer and suggests a potential critical pulmonary situation in the lung following epithelial exposure to MWCNT. Also required for the integrity of the vascular endothelial monolayer is the proper functioning of the endothelial junctions . Junctional contacts between endothelial cells act as semi-permeable barriers to allow the regulated passage of molecules between the vascular and interstitial spaces . One molecule that plays a key role in the maintenance of these junctions is VE-cadherin. VE-cadherin is restricted to endothelial cells and is a major component of intercellular junctions . Proper VE-cadherin function is necessary for development, and loss of VE-cadherin results in an increase in vascular permeability, thus permitting passage of fluid and macromolecules across the vascular barrier and resulting in inflammation and edema . The loss of the VE-cadherin signal at the cell surface of HMVEC after SAEC exposure to MWCNT, coupled with cell membrane ruffling and gap formation, suggests that the endothelial barrier had been compromised.
Exposure of SAEC to MWCNT also increased the angiogenic ability of the HMVEC cells. Physiological angiogenesis is a common occurrence in processes such as wound healing, which are self-limited in time, but pathological angiogenesis may occur unregulated for longer time periods . Chronic inflammatory diseases depend upon chronic neovascularization, and MWCNT have been shown to induce chronic inflammation with a shift to fibrosis in MWCNT-exposed mice [12, 14, 42]. Exposure of SAEC to MWCNT upregulates the angiogenic potential of HMVEC, suggesting that MWCNT exposure can induce the wound healing process and may potentially induce pathological angiogenesis. As no MWCNT were found to pass through the Transwell system into the endothelial well, cellular mediators secreted by the epithelial cells following MWCNT exposure appear to be the factor behind these endothelial cellular effects.
One of the main regulators of angiogenesis is VEGFA. VEGFA in the lung is released mainly by the epithelium, has a high specificity for endothelial cells, and is crucial for the maintenance of lung structure . VEGFA expression plays a role in endothelial cell survival, cell proliferation and migration, vasodilation, and enhanced vessel permeability . Expression of VEGFA is known to have a pathological role when dysregulated, and its increased expression plays a role in the development of inflammation, edema, and cancer . One mechanism behind increased endothelial cell permeability is VEGFA-induced endocytosis of VE-cadherin. As loss of VE-cadherin-mediated adhesion results in increased vascular permeability, VEGFA has the ability to induce an increase in vascular permeability through the loss of VE-cadherin signaling . The increase in angiogenic ability of HMVEC coupled with loss of VE-cadherin expression at the cell surface suggests that VEGFA may be playing a role in MWCNT-induced cellular signaling. Indeed, soluble VEGFA protein levels were shown to increase in the Transwell system following SAEC exposure to MWCNT, and this increase in expression correlated with increased angiogenesis and enhanced vascular permeability in HMVEC.
In addition to VEGFA, we chose to determine the levels of soluble intercellular adhesion markers, sICAM1 and sVCAM1, to determine their expression following SAEC exposure to MWCNT. ICAM1 and VCAM1, and their soluble forms, support the interaction of leukocytes with endothelial cells and are necessary for their transmigration . sICAM-1 and sVCAM-1 play a role in the development and severity of various diseases, such as cardiovascular disease, cancer, and autoimmune diseases [56, 57]. Both sICAM-1 and sVCAM-1 have been suggested to play a role in the upregulation of intracellular inflammatory mediators and endothelial dysfuction, resulting in endothelial cell migration, tube formation, and increased angiogenesis . Detection of sICAM-1 and sVCAM-1 in patients may provide a marker for the severity of disease and potential disease outcome. As the soluble cellular mediators in our system have the ability to pass back and forth through the Transwell membrane, it is not possible at this time to determine from which cell type the mediator originated. However, an overall increase of these soluble mediators is seen in the co-culture system, suggesting that cell-cell communication can occur between the epithelial and endothelial cells. Intracellular inflammatory signals involved in key regulatory pathways of inflammation were also assayed in HMVEC to determine innate changes within the cells. Phosphorylation of NF-κB and STAT3 is known to induce gene activation in various processes, such as proliferation, transformation, and apoptosis [58, 59]. NF-κB has been suggested to be a master regulator of inflammation, and its role is essential in the coordinated response to lung injury . NF-κB activation in endothelial cells has been shown to increase vascular permeability as well as upregulate the expression of cellular adhesion molecules, such as ICAM1 and VCAM1 . The activation of STAT3 in HMVEC following SAEC exposure to MWCNTs suggests a reaction to an induced inflammatory response and maintenance of cellular homeostasis . Sustained activation of STAT3 has been suggested to play a critical role in the pathogenesis of lung fibrosis by differentially inducing both apoptotic and proliferative cellular signals . Phosphorylation of the serine/threonine mitogen-activated protein kinase p38 occurs in response to inflammatory signals and results in the activation of proinflammatory mediators . p38 activation has been shown to regulate the expression of ICAM1 and VCAM1  and induce cytoskeletal remodeling in endothelial cells . Upregulated phosphorylation of the HMVEC intracellular inflammatory signals NF-κB, STAT3, and p38 MAPK in the co-culture system following SAEC exposure to MWCNT suggests that cellular mediators from the epithelial cells following exposure are able to elicit a response from the endothelial cells, which in turn respond by expression of their own cellular signaling. This cross-talk between the two cell types is a benefit of co-culture over mono-culture in that cellular signaling pathways can be relayed back and forth between the cell types to elicit a response which is more realistic to the in vivo environment.
The proper evaluation of current and newly created nanomaterials requires an efficient and relevant model system. As mono-culture studies of nanomaterial exposure have resulted in discordant results from those seen during in vivo exposure, we chose to develop a co-culture system in which to test the toxicity of epithelial exposure to MWCNT on the vascular endothelium in a relevant manner. A correlation study of global mRNA and miRNA expression between MWCNT exposure in SAEC and HMVEC mono-culture separately, SAEC and HMVEC in co-culture, and mouse lungs exposed to MWCNT determined that changes in gene expression due to MWCNT exposure in co-culture were highly correlated with those seen in vivo, while mono-culture exposure did not correlate well with the in vivo results (Snyder-Talkington, in preparation). Therefore, our co-culture system was a relevant model in which to test the potential lung toxicity of MWCNT to the vascular endothelium. Using this co-culture model, we determined that epithelial exposure to MWCNT induced multiple changes to the underlying endothelium, potentially through cell signaling mediators derived from MWCNT-exposed epithelial cells.