VEGF (vascular endothelial growth factor) overproduction has been identified as a major factor underlying pathological angiogenesis in vivo, including such conditions as psoriasis, macular degeneration, and tumor proliferation. Endothelial cell tyrosine kinase receptors, KDR and Flt-1, have been implicated in VEGF responses including cellular migration, proliferation, and modulation of vascular permeability. Therefore, agents that limit VEGF-cellular interaction are likely therapeutic candidates for VEGF-mediated disease states (particularly agents blocking activity of VEGF165, the most frequently occurring VEGF isoform). To that end, a nuclease-resistant, VEGF165-specific aptamer NX1838 (2'-fluoropyrimidine, RNA-based oligonucleotide/40-kDa-PEG) was developed. We have assessed NX1838 inhibition of a variety of cellular events associated with VEGF, including cellular binding, signal transduction, calcium mobilization, and induction of cellular proliferation. Our data indicate that NX1838 inhibits binding of VEGF to HUVECs (human umbilical vein endothelial cells) and dose-dependently prevents VEGF-mediated phosphorylation of KDR and PLCgamma, calcium flux, and ultimately VEGF-induced cell proliferation. NX1838-inhibition of VEGF-mediated cellular events was comparable to that observed with anti-VEGF monoclonal antibody, but was ineffective as an inhibitor of VEGF121-induced HUVEC proliferation. These findings, coupled with nuclease stability of the molecule, suggest that NX1838 may provide therapeutic utility in vivo.

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Thromboxane (Tx) A2 generation and subsequent selective pulmonary sequestration of neutrophils (PMNs) is characteristic of several forms of the adult respiratory distress syndrome (ARDS). Therefore, we examined PMN-dependent adhesion to cultured pulmonary microvessel and aortic endothelium (EC) in response to U46,619 (Tx mimic). Nonstimulated PMNs were two fold more adherent to pulmonary microvessel EC than to aortic EC (P less than 0.01). PMN pretreatment with Tx mimic (10(-6) M) increased adhesion to both types of EC (P less than 0.01). The Tx mimic-induced adhesion was blocked by receptor antagonists to Tx (SQ29,548) and to leukotrienes (FPL55,712), and by the anti-CD18 mAb TS1/18 (P less than 0.01, all cases). Baseline PMN adhesion also was modulated by Tx, leukotrienes, and CD18, for both EC types. These results indicate pulmonary microvessel EC is intrinsically more adhesive for both nonstimulated and stimulated PMNs than aortic EC and that Tx mediates PMN-dependent adhesion by coupled interaction of Tx and LT receptors via CD18 activation.

Anthracyclines, including daunorubicin (DnR) and doxorubicin (DoX), have shown clinical chemotherapeutic utility, albeit in association with cumulative dose-associated cardiotoxicities. Despite structural similarity, however, DnR and DoX treatments have been directed toward leukemias and solid tumor types, respectively. Due to a paucity of in vitro data regarding differential use of DnR or DoX, we assessed the cytotoxicity of these compounds against solid and hematological tumor cell types. In addition, we examined liposomal formulations of DnR (L-DnR) and DoX (PEG-DoX), which, in contrast to DnR or DoX, demonstrate antineoplastic activity with reduced cardiotoxicity in vivo. Accordingly, cytotoxicity testing (with [methyl/-(3)H]thymidine incorporation) of DnR, DoX, L-DnR, and PEG-DoX on a range of different human tumor cell lines (e.g., breast, lung, ovarian, prostate, melanoma, lymphoma, and leukemia tumor cell types) was performed. Our data indicate comparable activity for DnR, DoX, or L-DnR in all tumor cell types examined [e.g., SK-BR-3 (breast adenocarcinoma) cells: IC50 values = 5.9, 9.1, and 4.7 ng/mL for DnR, DoX, and L-DnR respectively]. In addition, several solid tumor cell types were more responsive to DnR than DoX [e.g., DU-145 (prostate carcinoma) cells: IC50 values = 10.4 and 41.2 ng/mL for DnR and DoX, respectively; p >. 001]. Interestingly, PEG-DoX was substantively less effective for all tumor cells (IC50 values were about 100-10,000 times greater for PEG-DoX than for DnR, DoX, or L-DnR; p >. 001, all cases). Reduced PEG-DoX activity in vitro may be related to polyethylene glycol (PEG) moieties present on the liposomal exterior of PEG-DoX, which are not present on L-DnR. Nonetheless, taken together, these data suggest that DnR and DoX demonstrate comparable efficacy in vitro and that specific liposomal encapsulation (L-DnR) does not mitigate DnR efficacy in vitro.

We previously demonstrated that the f-actin cytoskeleton modulates oxygen radical production associated with polymorphonuclear leukocyte (PMN) oxidative burst activity. Given the close association of the actin and microtubule cytoskeletons with the plasma membrane and the transmembrane location of the PMN NADPH oxidase, it is likely cytoskeletal change may affect PMN membrane responses, such as cellular anisotropy. Changes in PMN membrane fluidity were therefore examined after PMN activation by the chemoattractant N-formyl-1-methionyl-1-leucyl-1-phenylalanine (fMLP) in the presence or absence of phalloidin or cytochalasin B (CB), agents that stabilize and disrupt f-actin, or taxol and vincristine, which stabilize and disrupt microtubules, respectively. Phalloidin and taxol treatment of PMN significantly decreased whereas CB and vincristine significantly increased membrane fluidity. Activation of PMN by fMLP (10(-6) M) resulted in a significant increase in membrane fluidity that was attenuated by PMN pretreatment with phalloidin or taxol. CB and vincristine pretreatment of PMN did not alter the fMLP response. These data suggest that stabilization of the f-actin or microtubule cytoskeleton may prevent increases in cellular membrane fluidity associated with PMN activation.

Polymorphonuclear leukocyte (PMN) sequestration within the pulmonary microvasculature is known to occur in association with ischemia/reoxygenation (I/R). This sequestration is dependent on eicosanoids and reactive oxygen species. PMN sequestration within the lungs suggests that pulmonary microvascular endothelial cells (MECs) may in part regulate the I/R response. Simulating I/R, we examined the effect of hypoxia/reoxygenation (H/R) on pulmonary MECs in vitro, with and without PMNs. Significant cellular injury, assessed by 51Cr release, occurred upon reoxygenation of MECs (P < .01). Addition of PMNs to the H/R-injured monolayers did not increase MEC injury. Reoxygenation of MECs also resulted in increased thromboxane (Tx) B2 production compared to controls (P < .01). Inhibition of Tx secretion by aspirin reduced H/R-induced PMN adhesion to MECs (P < .01). Furthermore, H/R-induced increases in PMN-MEC adhesion were prevented by allopurinol and superoxide dismutase (P < .01). These data suggest that the pulmonary response to H/R is mediated by MEC generation of reactive oxygen radical species and Tx, which promotes increased PMN adhesion.

Re-expansion of atelectatic lung is associated with increased permeability. This study tests whether neutrophils mediate this event. Right middle lobar atelectasis was induced in anesthesized rabbits (n = 18) by intraluminal obstruction of the bronchus after a 20-minute ventilation with 100% O2. After 1 hour of bronchial obstruction and 20 minutes after lobar re-expansion, leukopenia was noted, 2870 +/- 210 white blood cells (WBC)/mm3, relative to control animals treated with a noninflated balloon catheter, 6500 +/- 410 WBC/mm3 (p less than 0.05). Three hours after re-expansion, neutrophils were sequestered in the previously atelectatic region 78 +/- 7 polymorphonuclear leukocytes (PMN)/10 high-power field (HPF), as well as in nonatelectatic areas, 40 +/- 3 PMN/10 HPF, higher than control values of 26 +/- 3 PMN/10 HPF (p less than 0.05). In the atelectatic region, neutrophil sequestration was associated with increased protein concentration in lobar bronchoalveolar lavage (BAL) of 1370 +/- 100 micrograms/mL, higher than control values of 270 +/- 20 micrograms/mL (p less than 0.05). Reexpansion also induced increases in lung wet-to-dry weight ratio (W/d) of 6.2 +/- 0.2, higher than control values of 4.3 +/- 0.1 (p less than 0.05). Rendering rabbits neutropenic (n = 18) (0 to 4 PMN/mm3) limited the atelectasis-induced protein accumulations in BAL (520 +/- 60 micrograms/mL) and increase in lung W/d (5.2 +/- 0.1) (both p less than 0.05). Intravenous (I.V.; treatment of another group (n = 18) with an anti-CD 18 monoclonal antibody (R 15.7, 1 mg/kg) before balloon deflation prevented leukopenia (6550 +/- 560 WBC/mm3), minimized neutrophil sequestration (36 +/- 2 PMN/10 HPF), and attenuated protein leak (710 +/- 95 micrograms/mL) and the increased lung W/d (5.6 +/- 0.1) (all p less than 0.05). A final atelectatic group (n = 9) was treated I.V. with the anti-intercellular adhesion molecule-1 monoclonal antibody (RR 1/1, 1 mg/kg), which also prevented leukopenia and showed similar protection of microvascular barrier function. These data indicate that adherent neutrophils in large part mediate lung permeability and edema after atelectasis and re-expansion. Adhesion receptors of both neutrophils and endothelial cells regulate this event.

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