MN10021 inhibits dermal vascular leak in a reverse passive Arthus reaction.

<p>Groups of 5 Hartley strain guinea pigs (male; ∼250 g) were injected s.c. with 1.0 ml of either saline or the indicated dose of MN10021. Eighteen hours later the flanks of the animals were shaved and both 6.25 and 12.5 µl of rabbit anti-BSA antiserum injected i.d. at each of two sites, one on each flank. The animals were then injected i.v. with 1.0 ml of PBS containing BSA (1.0 mg/ml) and Evans Blue dye (2.0 mg/ml). Six hours later the animals were euthanized by barbiturate overdose, the skin on the back removed, and the diameter of the extravasation of the Evans Blue dye measured for each site on the ventral surface of the skin. The values represent the mean ± SE of the 10 measurements obtained for each of the treatment conditions. ** p<0.01; * p<0.05; NS  =  not significant.</p

MN10021 and DUK0005 inhibit apoptosis and necrosis in HUVEC.

<p>A. HUVEC were grown to confluence in 96-well tissue culture plates. Media was removed from the wells and to each of quadruplicate wells was added 200 µl of either fresh media (untreated) or fresh media containing 50 µM MN10021 (treated) and the cells incubated 3 h at 37°C. To quadruplicate wells of both untreated and treated cells were added 20 µl of media or 20 µl of media containing either 100 or 500 nM staurosporine. The plates were incubated overnight at 37°C and the cells processed for measurement of apoptosis using the Cell Death Detection assay (Roche Diagnostics) per the manufacturer's instructions. The apoptotic index was calculated as described in Methods. B. HUVEC were grown to confluence in 96-well tissue culture plates. Media was removed from the wells and to each of quadruplicate wells was added 200 µl of either fresh media (untreated) or fresh media containing 50, 25, 12.5, or 6.25 µM MN10021 (treated) and the cells incubated 3 h at 37°C. To quadruplicate wells of both untreated and treated cells were added 20 µl of media containing 100 ng/ml human TNF-α (recombinant; R & D Systems). The plates were incubated overnight at 37°C and the supernatants removed and processed for measurement of necrosis using the Cell Death Detection assay (Roche Diagnostics) per the manufacturer's instructions. Absorbance values for the supernatants of untreated cells (no TNF-α) were negligible. C. HUVEC were grown to confluence in 96-well tissue culture plates. Media was removed from the wells and to each of quadruplicate wells was added 200 µl of either fresh media (untreated) or fresh media containing 50, 25, 12.5 µM DUK0005 (treated) and the cells incubated 3 h at 37°C. To quadruplicate wells of both untreated and treated cells were added 20 µl of media or 20 µl of media containing 100 nM staurosporine. The plates were incubated overnight at 37°C and the cells processed for measurement of apoptosis using the Cell Death Detection assay (Roche Diagnostics) per the manufacturer's instructions. Absorbance values for untreated cells (no staurosporine) were negligible. *** p<0.001; ** p<0.01; * p<0.05.</p

Effect of MN10021 on blood leukocyte and platelet counts in murine DIC model.

<p>Each of 4 groups of 5 CD-1 mice (male, 6–8 weeks of age) was injected i.p with 1.0 ml of either saline (closed circles) or 1.0 mg (245 nmol) MN10021 in saline (open circles). Eighteen hours later all mice were injected i.p. with 1.0 ml of 0.45% carrageenan. At each of the indicated times one group of 5 mice for each treatment was euthanized, an anti-coagulated blood sample obtained by cardiac puncture, and total white blood cell (WBC) (panel A) or platelet (PLT) (panel B) counts performed on an automated hematology analyzer. In both panels the dotted line represents the average value obtained from the blood samples of 10 untreated CD-1 mice. The data plotted are the mean values ± SE. Error bars were calculated for all values and if not visible are contained within the symbol.</p

Inhibition of apoptosis in HUVEC correlates with production of NO by retroviral-derived peptides.

<p>A. HUVEC were grown to 70–80% confluence in 96-well tissue culture plates. Media was removed from the wells and to each of quadruplicate wells was added 200 µl of either fresh media (untreated) or fresh media containing 50 µM of the indicated peptides (treated) and the cells incubated ON at 37°C. Supernatant was removed from each well as assayed for total NO by measurement of nitrite with a Colorimetric Nitric Oxide Assay Kit (Oxford Biomedical Research; Oxford, MI) which employs nitrate reductase to convert nitrate to nitrite. B. HUVEC were grown to confluence in 96-well tissue culture plates. Media was removed from the wells and to each of quadruplicate wells was added 200 µl of either fresh media (untreated) or fresh media containing 50 µM of the indicated peptide (treated) and the cells incubated 3 h at 37°C. To quadruplicate wells of both untreated and treated cells were added 20 µl of media or 20 µl of media containing 500 nM staurosporine. The plates were incubated overnight at 37°C and the cells processed for measurement of apoptosis using the Cell Death Detection Assay (Roche Diagnostics) per the manufacturer's instructions.</p

Pretreatment with L-NAME does not affect peptide inhibition of apoptosis.

<p>HUVEC were grown to 70–80% confluence in a 96-well tissue culture plate. Media was removed from the wells and to each of quadruplicate wells was added 200 µl of either fresh media (untreated) or fresh media containing 2 mM L-NAME and the cells incubated 5 h at 37°C. To each well was then added the indicated peptide to achieve a concentration of 50 µM and the cells incubated an additional 3 h at 37°C before addition of staurosporine to a final concentration of 500 nM. The plate was incubated ON at 37°C and the cells processed for measurement of apoptosis using the Cell Death Detection Assay (Roche Diagnostics) per the manufacturer's instructions.</p

Anti-Inflammatory and Vasoprotective Activity of a Retroviral-Derived Peptide, Homologous to Human Endogenous Retroviruses: Endothelial Cell Effects

<div><p>Malignant and inflammatory tissues sometimes express endogenous retroviruses or their proteins. A highly-conserved sequence from retroviral transmembrane (TM) proteins, termed the “immunosuppressive domain (ID)”, is associated with inhibition of immune and inflammatory functions. An octadecapeptide (MN10021) from the ID of retroviral TM protein p15E inhibits <em>in vitro</em> release of pro-inflammatory cytokines and increases synthesis of anti-inflammatory IL-10. We sought to determine if MN10021 has significant <em>in vivo</em> effects. MN10021, prepared by solid-phase synthesis, was dimerized through a naturally-occurring, carboxy-terminal cysteine. <em>In vivo</em> anti-inflammatory activity was determined using a murine model of sodium periodate (NaIO₄)-induced peritonitis. <em>In vivo</em> vasoprotective effects were determined using: (1) a carrageenan-induced model of disseminated intravascular coagulation (DIC) in mice; (2) a reverse passive Arthus model in guinea pigs; and (3) vasoregulatory effects in spontaneously hypertensive rats (SHR). <em>In vitro</em> studies included: (1) binding/uptake of MN10021 using human monocytes, cultured fibroblasts, and vascular endothelial cells (VEC); (2) gene expression by RT-PCR of MN10021-treated VEC; and (3) apoptosis of MN10021-treated VEC exposed to staurosporine or TNF-α. One-tenth nmol MN10021 inhibits 50 percent of the inflammatory response in the mouse peritonitis model. Furthermore, 73 nmol MN10021 completely protects mice in a lethal model of carrageenan-induced DIC and inhibits vascular leak in both the mouse DIC model and a guinea pig reverse passive Arthus reaction. MN10021 binds to and is taken up in a specific manner by both human monocytes and VEC but not by cultured human fibroblasts. Surprisingly, orally-administered MN10021 lowers blood pressure in SHR rats by 10–15% within 1 h suggesting a direct or indirect effect on the vascular endothelium. MN10021 and derived octapeptides induce iNOS (inducible nitric oxide synthase) mRNA in VEC and nitrate in VEC cell culture supernatants and protect VEC from induced apoptosis or necrosis. However, pretreatment of VEC with nitro-L-arginine methyl ester (L-NAME), while inhibiting the release of nitrate, does not block the anti-apoptotic effect of MN10021 and derived octapeptides suggesting that their potent vasoprotective and anti-inflammatory activity is not nitric oxide dependent.</p> </div

Induction by MN10021 and analogs of mRNA for iNOS in HUVEC.

<p>A. HUVEC were grown to 80–90% confluence in 12-well tissue culture plates using defined media provided by Cambrex and supplemented with 10% FBS. The cells were allowed to become quiescent by incubating them overnight in the basal media (without growth factors) supplied by Cambrex and supplemented with 1% FBS. Triplicate wells were incubated with either media alone or media containing 50 µM MN10021 for 3 h at 37°C, the wells washed with warm PBS, and the cells lysed. Total RNA was isolated, cDNA prepared, and RT-PCR performed as described in Materials & Methods. Primers for human iNOS and β-actin were obtained from R & D systems. B. HUVEC were prepared as described under Methods, grown to 80–90% confluence in 12-well tissue culture plates, and incubated overnight in basal medium (no growth factors) containing 1% FBS in order to make them quiescent. Triplicate wells were incubated for 3 h at 37°C with 50 µM of one of four different 8-amino acid long analogs of MN10021. DUK0001: NH₂-GLDLLFLK-COOH; DUK0004: acetyl-GLDLLFLK-acetyl; DUK0005: acetyl-GLDLLFLK-NH₂; DUK0006: NH₂-GLDLLFLK-NH₂. The wells were washed with warm PBS, and the cells lysed. Total RNA was isolated, cDNA prepared, and RT-PCR performed as described in Materials & Methods. Primers for human iNOS and β-actin were obtained from R & D systems. C. HUVEC were prepared and tested as described in panel A. The sequence of DUK0007 is acetyl-GLDLLYLK-NH₂ which differs from that of DUK0005 in that it has a Tyr at position 6 in place of a Phe.</p

Effect of retroviral peptides on lethality associated with carrageenan-induced DIC in mice.

<p>A. Groups of 10 CD-1 mice (male, 6–8 weeks of age) were injected i.p. with 0.25 ml of PBS containing either 1 mg of either MN10021 or MN20050. Eighteen hours later the mice were injected i.p. with 1 ml of 0.45% carrageenan and survival was monitored over 48 h. The results shown are representative of >5 independent studies. B. Groups of 10 CD-1 mice (male, 6–8 weeks of age) were injected i.p. with the indicated doses of MN10021. Eighteen hours later the mice were injected i.p. with 1 ml of 0.45% carrageenan and survival was monitored over 48 h.</p

Monocyte and endothelial cell binding of MN10021 and MN20054.

<p>A. Human blood monocytes, isolated as described in Methods, were adjusted to 5×10⁶/ml in PBS (0.5% BSA, pH 7.5). Two-tenths ml of cell suspension was added to each of triplicate 12×75 mm polypropylene tubes and the tubes were incubated for 60 min at 37°C with 20 pmol of [¹²⁵I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, a 100-fold molar excess of MN10021, or a 100-fold molar excess of MN20050. The contents of each tube were then layered onto 0.8 ml of 10% sucrose in Eppendorf tubes, centrifuged, and the cell-associated radioactivity determined by gamma scintillation spectrophotometry of the cell pellet. B. Binding/uptake of [¹²⁵I]-MN20054 by human monocytes was performed as described for panel A with the following exceptions: (1) half the cells were pretreated for 30 min at 37°C with cytochalasin B to inhibit endocytosis and (2) competition was performed only with a 100-fold molar excess of MN10021. C. HUVEC were grown to confluence in 12-well tissue culture plates. To each of triplicate wells was added 20 pmol of [¹²⁵I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, a 100-fold molar excess of MN10021, or a 100-fold molar excess of MN20050. The plates were incubated for 60 min at 37°C with gentle rocking, the wells aspirated and quickly washed 3X with 2.0 ml each of ice-cold PBS/BSA and 1.0 ml of 1.0 N NaOH added to each well to solubilize the cells. Nine-tenths ml of solubilized cells was then removed from each well and cell-associated radioactivity determined by gamma scintillation spectrophotometry.</p

Potency of MN10021 in Mouse Peritonitis Model of Inflammation.

<p>ND  =  Not Determined.</p><p>NE  =  No Effect.</p><p>NA  =  Not Applicable.</p