FLX treatment enhances atherogenesis by promoting leukocyte recruitment

Chronic FLX administration is known to deplete intra-platelet 5-HT, which reflects the major peripheral 5-HT storage site. This has been reported in depressed patients treated with SSRIs as well as in mice receiving the same drug.^115,131 Indeed, after seven days of FLX administration, mice possessed a significant decrease in serum 5-HT. Because several studies associate platelet 5-HT with a pro-inflammatory role in diseases such as colitis,⁸² asthma⁸³, inflammatory bowel disease,⁸² and obesity,⁸⁴ the pro-atherogenic effect of FLX in atherogenesis was surprising. Herr et al. described that an acute FLX treatment leads to transiently elevated plasma 5-HT levels within the first hours, thereby promoting leukocyte-endothelial interactions.¹¹⁶ However, the same pro-atherogenic effect of FLX was observed irrespective of the time point of FLX onset, with or without FLX pretreatment for 2 weeks before starting the atherogenic diet. Thus, temporary increased 5-HT concentrations were excluded as underlying cause for the observed increase in plaque formation. FLX administration neither altered the plaque composition in advanced lesions, nor the progression of already established plaques. Remarkably, the FLX-mediated increase in lesion size was most pronounced during the early phase of atherogenesis. These findings suggest that FLX may affect the processes involved in plaque formation such as myeloid cell recruitment, enhanced platelet activity and endothelial activation. Indeed, FLX-treated mice exhibited an increased neointimal macrophage accumulation. This might be caused by enhanced transendothelial migration, since FLX-treated mice revealed an augmented vascular permeability and increased arterial adhesion of myeloid cells.

As already mentioned above, myeloid cell recruitment to the arterial wall is a crucial step in plaque formation. The interaction of circulating leukocytes with the endothelium involves a coordinated interplay of endothelial adhesion molecules with their counterparts on leukocytes.²⁴ On the part of the endothelium, an FLX-induced modulation of the adhesion molecules was excluded. This contradicts previously published in vitro data, which reported that FLX decreased TNFα-induced ICAM-1 and VCAM-1 protein levels in human aortic endothelial cells.¹⁸³ However, apart from direct in vitro stimulation of murine immortalized endothelial cells, the observation that FLX does not affect endothelial adhesion molecules was confirmed by mRNA and protein measurement in aortas of FLX-treated mice. This provides evidence that FLX is not altering protein levels of adhesion molecules in these settings. Cell transmigration is also dependent on the vascular integrity, which can be regulated and maintained by platelets.⁵⁶ 5-HT is known to provoke platelet activity by a positive feedback mechanism leading to amplified platelet activation.¹⁰⁶ One may speculate that the detected increase in vascular permeability upon FLX-treatment is caused by reduced platelet activation.

However, platelet characteristics were similar between control and FLX-treated mice. Thus, the aggravated endothelial permeability might be rather a secondary consequence of FLX-mediated leukocyte recruitment. The barrier function of the endothelium is not only maintained

by platelets but also by multiple proteins regulating the intercellular junctions between endothelial cells. By altering the strength of the endothelial cell-cell interactions, the organism can adjust vascular permeability to adapt to particular needs. In addition, endothelial permeability is increased by inflammatory stimulation. Several proteins are involved in the regulation of permeability of which claudins, occludins and JAMs are the best known.⁶ It is conceivable that FLX affects endothelial intercellular junctions, which was not further investigated in this study.

Lesional leukocytes originate from bone marrow and spleen, from where they are recruited into the circulation and subsequently to the vessel wall. An effect of FLX on these organs was excluded, based on the observation that similar myeloid cell counts in these organs were determined in FLX treated mice compared to the control group. The observed transient decrease in circulating leukocyte and platelet counts might be a consequence of enhanced arterial adhesion. In humans and mice, the increased frequency of circulating PLAs is associated with more severe inflammation.^61,63 However, FLX-treated mice did not reveal a higher incidence of PLAs, which could be one explanation for decreased platelet number and amplified adhesion. The detected neutropenia and enhanced endothelial recruitment is somehow in conflict with previously published data obtained in mice lacking peripheral 5-HT.¹¹⁵ Duerschmied and colleagues observed a mild neutrophilia and less neutrophil adhesion to mesenteric post-capillary venules in mice deficient for TPH1 as well as in wild type mice treated with FLX.¹¹⁵ The opposing findings might arise from the different vascular beds examined by Duerschmied et al. and the present study. While Duerschmied et al. assessed leukocyte adhesion in the microvasculature, the present study performed intravital microscopy of atherosclerosis-prone carotid arteries, which refers to macrovasculature. In large arteries under high blood pressure, leukocyte recruitment to the endothelium may require other mechanisms than those described in post-capillary venules to facilitate adhesion.³³ Indeed, dissimilarities in leukocyte adhesion between micro- and microvasculature were observed before.^17,18 Drechsler et al. found that recruitment of neutrophils to large arteries is dependent on the chemokine receptors CCR1, CCR2, CCR5, and CXCR2, whereas recruitment to peripheral veins is independent of CCR1 and CCR5 and only requires CCR2 and CXCR2.

They propose that this is ascribed to the deposition of the CCR1 and CCR5 ligand CCL5 on the endothelium by platelets, which occurs in arteries but not in veins.¹⁷ Similarly, Ortega-Gomez et al. demonstrated that neutrophil-derived cathepsin G preferentially binds to arterial rather than venular endothelium in a CCL5-dependent manner, thereby inducing integrin clustering, which in turn results in arterial-specific myeloid cell adhesion.¹⁸ They speculate that differences in the composition of surface molecules on the arterial endothelium are causing these observations.¹⁸ In fact, shear forces are much higher in macrocirculation compared to microcirculation. As a consequence, the endothelium and the mechanisms involved in

leukocyte adhesion are adapted to withstand those high flow rates. Scott et al. highlighted the heterogeneity that exists on the endothelium from different vascular beds. For example, ICAM1 and VCAM1 expression patterns upon inflammatory stimulation differ between endothelial cells from different origins. Furthermore, they also found that N-glycan profiles on the cell surfaces are vascular bed-specific,¹⁸⁴ which may explain the vessel-dependent dissimilarities in CCL5 binding.¹⁸ Thus, site-specific differences in this study (macrovasculature) compared to the one from Duerschmied et al. (microvasculature) may explain the opposing findings. Another important aspect is the underlying hypercholesterolemia in ApoE^-/- mice receiving a HFD.

These mice display a chronic inflammation including endothelial dysfunction. The relevance of the inflammatory setting and the site-specificity is corroborated by the findings that non-atherogenic wild type mice treated with FLX did not exhibit the transient reduction in blood counts and showed less neutrophil extravasation in an acute peritonitis model.

The recruitment of leukocyte to the site of inflammation is not only dependent on alterations of the endothelium but also on the activation of leukocytes.²⁴ The leukocyte-endothelial binding is mediated by the interaction of selectins and integrins with their corresponding ligands. FLX treatment neither altered the surface levels of adhesion molecules such as PSGL1 and CD62L nor the protein levels of several integrins on circulating neutrophils. The in vivo imaging of leukocyte adhesion revealed that FLX affects adhesion but not rolling. The main adhesion proteins involved in this process are the integrins. These molecules are rather regulated by activation than changes in expression. Several studies described that integrin activation and clustering are important events in atherogenesis.^18,161 Indeed, neutrophils and monocytes of FLX-treated mice revealed an increased CCL5-induced integrin activity, which was observed in both β1- and β2-integrins. Remarkably, in vitro stimulation of blood leukocytes of wild type mice and human neutrophils-like cells with FLX amplified CCL5-induced integrin binding capacity. However, this effect only occurred in the presence of CCL5. This positively supports the hypothesis that the observed FLX-mediated pro-atherogenic effect is site-specific and dependent on the presence of enhanced chemokine levels, as in the case of chronic hypercholesterolemia. The fact that 5-HT in vitro stimulation of mouse and human cells did not affect integrin capability provides evidence that the in vivo detected pro-atherogenic phenotype of FLX is independent of 5-HT platelet depletion.