To extend our in vitro findings that the FTY720 analogs promote lung EC integrity, we employed a well characterized murine model of LPS-induced lung injury (Materials and Methods) to examine the in vivo effects of these compounds on pulmonary vascular leak and inflammatory injury. Preliminary studies indicated that 1S was superior to 3-deazaneplanocin A (DZNeP) HCl the other barrier-promoting analogs (1R, 2R, and 2S) in this model (data not shown). Therefore, we proceeded to further characterize the representative barrier-enhancing FTY720 analog 1S on pulmonary vascular leak and inflammatory injury in this mouse model. As we have described previously (Peng et al., 2004), intratracheal administration of LPS (2.5 mg/kg) produces significant murine inflammatory lung injury at 18 h as assessed by measurements of BAL total protein and cell count, BAL albumin, and lung tissue albumin.

Moreover, LPS increases tissue MPO activity, another reflection of lung parenchymal phagocyte infiltration, compared with control mice (Peng et al., 2004). Intraperitoneal injection of a single dose of FTY720 analog 1S (0.1�C5.0 mg/kg) delivered 1 h after LPS exposure significantly reduces capillary leak relative to PBS control at all of the concentrations studied as measured by total BAL protein concentrations (Fig. 6A). This reduction in permeability by 1S is comparable to that achieved by S1P or FTY720. In addition, 1S significantly reduces LPS-induced albumin leakage from the vascular space into both the surrounding lung tissue and BAL (Fig. 6, B and C), as well as BAL WBC accumulation and lung tissue MPO activity (Fig.

7, A and B). These combined data suggest that the optimal protective dose of 1S is 0.1 to 1.0 mg/kg in this model. Fig. 6. FTY720 analog 1S reduces LPS-induced vascular permeability in murine lungs. A, male C57BL/6 mice were given LPS (2.5 mg/kg) intratracheally. One hour later, mice received PBS vehicle, FTY720 (0.5 mg/kg), or 1S (doses labeled on the graph, milligram/kilogram) … Fig. 7. 1S reduces LPS-induced WBC accumulation in mouse lungs. A, in mice treated as described in Fig. 6, BAL fluid was also analyzed for total WBC count. n = 3�C5 animals per condition. , p < 0.05, , p < 0.01, ... One potential concern when using FTY720 or related compounds in sepsis-related processes such as acute lung injury is the known lymphopenia effect of the parent compound (Kovarik et al.

, 2004). Therefore, peripheral blood WBC levels were assessed in this mouse model. For comparison, at baseline Brefeldin_A in control mice (no LPS), total circulating WBC is 4.11 �� 1.58 �� 103/��l and the lymphocyte count is 3.57 �� 1.74 �� 103/��l (n = 6), so these levels are significantly suppressed (p �� 0.001 for both total WBC and lymphocyte count) by LPS alone in this model 18 h after its administration (Fig. 8).