Vasoprotective effects of human CD34+ cells: towards clinical applications

<p>Abstract</p> <p>Background</p> <p>The development of cell-based therapeutics for humans requires preclinical testing in animal models. The use of autologous animal products fails to address the efficacy of similar products derived from humans. We used a novel immunodeficient rat carotid injury model in order to determine whether human cells could improve vascular remodelling following acute injury.</p> <p>Methods</p> <p>Human CD34+ cells were separated from peripheral buffy coats using automatic magnetic cell separation. Carotid arterial injury was performed in male Sprague-Dawley nude rats using a 2F Fogarty balloon catheter. Freshly harvested CD34+ cells or saline alone was administered locally for 20 minutes by endoluminal instillation. Structural and functional analysis of the arteries was performed 28 days later.</p> <p>Results</p> <p>Morphometric analysis demonstrated that human CD34+ cell delivery was associated with a significant reduction in intimal formation 4 weeks following balloon injury as compared with saline (I/M ratio 0.79 ± 0.18, and 1.71 ± 0.18 for CD34, and saline-treated vessels, respectively P < 0.05). Vasoreactivity studies showed that maximal relaxation of vessel rings from human CD34+ treated animals was significantly enhanced compared with saline-treated counterparts (74.1 ± 10.2, and 36.8 ± 12.1% relaxation for CD34+ cells and saline, respectively, P < 0.05)</p> <p>Conclusion</p> <p>Delivery of human CD34+ cells limits neointima formation and improves arterial reactivity after vascular injury. These studies advance the concept of cell delivery to effect vascular remodeling toward a potential human cellular product.</p

Tissue Factor Pathway Inhibitor Overexpression Inhibits Hypoxia-Induced Pulmonary Hypertension

Pulmonary hypertension (PH) is a commonly recognized complication of chronic respiratory disease. Enhanced vasoconstriction, pulmonary vascular remodeling, and in situ thrombosis contribute to the increased pulmonary vascular resistance observed in PH associated with hypoxic lung disease. The tissue factor pathway regulates fibrin deposition in response to acute and chronic vascular injury. We hypothesized that inhibition of the tissue factor pathway would result in attenuation of pathophysiologic parameters typically associated with hypoxia-induced PH. We tested this hypothesis using a chronic hypoxia–induced murine model of PH using mice that overexpress tissue factor pathway inhibitor (TFPI) via the smooth muscle–specific promoter SM22 (TFPISM22). TFPISM22 mice have increased pulmonary TFPI expression compared with wild-type (WT) mice. In WT mice, exposure to chronic hypoxia (28 d at 10% O2) resulted in increased systolic right ventricular and mean pulmonary arterial pressures, changes that were significantly reduced in TFPISM22 mice. Chronic hypoxia also resulted in significant pulmonary vascular muscularization in WT mice, which was significantly reduced in TFPISM22 mice. Given the pleiotropic effects of TFPI, autocrine and paracrine mechanisms for these hemodynamic effects were considered. TFPISM22 mice had less pulmonary fibrin deposition than WT mice at 3 days after exposure to hypoxia, which is consistent with the antithrombotic effects of TFPI. Additionally, TFPISM22 mice had a significant reduction in the number of proliferating (proliferating cell nuclear antigen positive) pulmonary vascular smooth muscle cells compared with WT mice, which is consistent with in vitro findings. These findings demonstrate that overexpression of TFPI results in improved hemodynamic performance and reduced pulmonary vascular remodeling in a murine model of hypoxia-induced PH. This improvement is in part due to the autocrine and paracrine effects of TFPI overexpression

Cell Surface Protein Disulfide Isomerase Regulates Natriuretic Peptide Generation of Cyclic Guanosine Monophosphate

<div><p>Rationale</p><p>The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated.</p><p>Objective</p><p>We utilized complementary <i>in vitro</i> and <i>in vivo</i> models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate.</p><p>Methods and Results</p><p>Inhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI <i>in vivo</i> attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry.</p><p>Conclusion</p><p>These data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.</p></div

Differential binding of NPs and PDI.

<p><b>A</b>. Binding between immobilized PDI and 200 nM NPs was analyzed by SPR. <b>B and C.</b> Surface plasmon resonance (SPR) signals when immobilized PDI protein was exposed to varying concentrations (0–1600 nM) of ANP and BNP (representative from 3 independent experiments). <b>D</b>. Dissociation constants for complexes of NPs with PDI protein. (Error bars, +SD of 3 independent experiments using different chips and peptide preparations.).</p

Down regulated PDI attenuates CNP-mediated cGMP activation in HMCs.

<p><b>A</b>. Western blotting with RL90 antibody shows partial knockdown of PDI in HMCs. <b>B</b>. Effect of PDI knockdown on cGMP activation. Data are expressed as mean<u>+</u>SEM vs control cells without PDI siRNA treatment (*P≤0.05). <b>C</b>. PDI inhibition by RL90 decreases ANP, BNP and CNP-mediated cGMP generation in HMC (*p≤0.05). (Error bars, +SD from 3 independent experiments, samples were triplicated in each experiment).</p

Bacitracin inhibited PDI attenuates NP-mediated generation of cGMP in vascular cells.

<p>Bacitracin (Ba) inhibits NPs activation in HMC (A), HVSMC (B) and HUVEC (C) * p ≤0.05 vs samples without bacitracin addition. (Error bars, +SD from 3 independent experiments, samples were triplicated in each experiment).</p

PDI inhibition attenuates NP-mediated generation of cGMP in vivo.

<p>Mice were given ANP with or without PDI inhibitors. Blood samples were collected, cGMP levels assessed. Data are expressed as mean<u>+</u>SEM (n = 6 mice). * p value ≤0.05 vs ANP with addition of RL90 antibody, and ** p value ≤0.001 with addition of Bacitracin.</p