Cell Membrane Modification for Rapid Display of Bi-Functional Peptides: A Novel Approach to Reduce Complement Activation

Ischemia and reperfusion of organs is an unavoidable consequence of transplantation. Inflammatory events associated with reperfusion injury are in part attributed to excessive complement activation. Systemic administration of complement inhibitors reduces reperfusion injury but leaves patients vulnerable to infection. Here, we report a novel therapeutic strategy that decorates cells with an anti-complement peptide. An analog of the C3 convertase inhibitor Compstatin (C) was synthesized with a hexahistidine (His6) tag to create C-His6. To decorate cell membranes with C-His6, fusogenic lipid vesicles (FLVs) were used to incorporate lipids with nickel (Ni2+) tethers into cell membranes, and these could then couple with C-His6. Ni2+ tether levels to display C-His6 were modulated by changing FLV formulation, FLV incubation time and FLV levels. SKOV-3 cells decorated with C-His6 effectively reduced complement deposition in a classical complement activation assay. We conclude that our therapeutic approach appears promising for local ex vivo treatment of transplanted organs to reduce complement-mediated reperfusion injury

Folic acid mitigates angiotensin-II-induced blood pressure and renal remodeling.

Clinical data suggests an association between systolic hypertension, renal function and hyperhomocysteinemia (HHcy). HHcy is a state of elevated plasma homocysteine (Hcy) levels and is known to cause vascular complications. In this study, we tested the hypothesis whether Ang II-induced hypertension increases plasma Hcy levels and contributes to renovascular remodeling. We also tested whether folic acid (FA) treatment reduces plasma Hcy levels by enhancing Hcy remethylation and thus mitigating renal remodeling. Hypertension was induced in WT mice by infusing Ang II using Alzet mini osmotic pumps. Blood pressure, Hcy level, renal vascular density, oxidative stress, inflammation and fibrosis markers, and angiogenic- and anti-angiogenic factors were measured. Ang II hypertension increased plasma Hcy levels and reduced renal cortical blood flow and microvascular density. Elevated Hcy in Ang II hypertension was associated with decreased 4, 5-Diaminofluorescein (DAF-2DA) staining suggesting impaired endothelial function. Increased expression of Nox-2, -4 and dihydroethidium stain revealed oxidative stress. Excess collagen IV deposition in the peri-glomerular area and increased MMP-2, and -9 expression and activity indicated renal remodeling. The mRNA and protein expression of asymmetric dimethylarginine (ADMA) was increased and eNOS protein was decreased suggesting the involvement of this pathway in Hcy mediated hypertension. Decreased expressions of VEGF and increased anti-angiogenic factors, angiostatin and endostatin indicated impaired vasculogenesis. FA treatment partially reduced hypertension by mitigating HHcy in Ang II-treated animals and alleviated pro-inflammatory, pro-fibrotic and anti-angiogenic factors. These results suggest that renovascular remodeling in Ang II-induced hypertension is, in part, due to HHcy

mRNA and protein expression of CBS/CSE and MTHFR is decreased in Ang II induced hypertension

<p>Effect of vehicle, Ang II and FA on the mRNA and protein expression of CBS (<b>A</b>); CSE (<b>C</b>) and MTHFR (<b>E</b>) as determined by semiquantitative RT-PCR and Western blotting. Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. Bar diagrams represent fold change from n = 6 experiments using GAPDH as control. * p<0.05 vs. vehicle; ^† p<0.05 vs. Ang II, ^‡ p<0.05 vs. vehicle.</p

Folic acid treatment increased renal cortical blood flow and vascular density in Ang II infused kidney.

<p>(<b>A</b>) Renal cortical blood flow was measured at end-point using Speckle contrast Imager (MoorFLPI, Wilmington, DE). Animals were anesthetized with TBE (Tribromoethanol, 240 mg/kg b.w. i.p.) and the left kidney exposed. All measurements were done under standard conditions of light and temperature control. (<b>B</b>) Data was first analyzed with ANOVA and pairwise comparison was performed using Bonferroni method. Summarized bar diagram represents mean ± SEM, n = 5–6 animals/group. * p<0.05 vs. vehicle and ^†p<0.05 vs. Ang II. (<b>C</b>) Mice were infused with Barium sulfate (100 mg/ml, at pH 5.0) through PE10 catheter (ID -0.28 MM, Franklin Lakes, NJ) inserted in the carotid artery directed towards the aorta and a constant rate of 200 µL/min was injected. Two minute X-ray images were captured with Kodak 4000 MM image station (Molecular Imaging System; Carestream Health Inc., Rochester, NY). Image analyses were done by ImagePro software (a representative analysis image is shown at the bottom right). Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. Bar diagram indicates percent change of vascular density against the background using vehicle treatment as control, n = 3 mice/group. * p<0.05 vs. vehicle; ^† p<0.05 vs. Ang II.</p

FA mitigated glomerulosclerosis and collagen IV expression in Ang II hypertension.

<p>(<b>A</b>) Histological kidney sections (5 µm thick) were stained with Masson's trichrome. Collagen is shown as deep blue color (yellow arrows); 200× magnification. (<b>B</b>) Collagen IV was detected in the cortical tissue extracted protein by Western blot. (<b>C</b>) Bar diagram represents densitometric analysis of collagen IV expression as fold change against vehicle group, n = 5–6 group. Statistical analyses were performed by Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. * p<0.05 vs. vehicle and, ^† p<0.05 vs. Ang II.</p

FA treatment reduces ROS production in Ang II hypertension by decreasing Nox-2 and Nox-4 isoforms.

<p>(<b>A</b>) Equal amount of protein from each group was separated on a SDS-PAGE and immunoblotted with anti-Nox-2, and -4 antibodies. (<b>B</b>) The pixel densities of bands from n = 6/group were quantified using ImageJ software (National Institute of Health, NIH) and presented as fold change using β-actin as control. Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. * p<0.05 vs. vehicle; p<0.05 vs. Ang II. (<b>C</b>) Superoxide was detected in the glomerulus by dihydroethidium (DHE) staining as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083813#s2" target="_blank">Materials and Methods</a>. Scale 20 µm.</p

HHcy in Ang II hypertension increases expression of ADMA and suppresses eNOS.

<p>Effect of vehicle, Ang II and FA on mRNA and protein expression of ADMA (<b>A</b>) and protein expression of eNOS (<b>C</b>) by RT-PCR and Western blotting. Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. Bar diagrams represent fold change for ADMA (<b>B</b>) and eNOS (<b>C</b>) from n = 6 animals/group. GAPDH was used as control. * p<0.05 vs. vehicle; ^† p<0.05 vs. Ang II, ^‡ p<0.05 vs. vehicle.</p

FA normalized altered expression of angiogenic and anti-angiogenic factors in Ang II hypertension.

<p>Western blot was performed to measure VEGF, angiostatin and endostatin expression using specific antibodies in the renal cortical tissue extracted protein. Statistical analyses were performed with Kruskal-Wallis test and individual pairs were compared using Mann-Whitney Rank sum test. Bar diagrams represent fold change from n = 5–6 mice/group. * p<0.05 vs. vehicle and ^† p<0.05 vs. Ang II, ^‡ p<0.05 vs. vehicle.</p

Food and water intake.

<p>Food and water intake was measured for 5 days. Vehicle group represents animals treated with normal saline; Ang II, angiotensin II; FA, folic acid. Data is presented as mean ± SEM. * p<0.01 vs. vehicle control; ^† p<0.05 vs. Ang II + FA.</p