David W. Courtman

Rescue of Monocrotaline-Induced Pulmonary Arterial Hypertension Using Bone Marrow–Derived Endothelial-Like Progenitor Cells Efficacy of Combined Cell and eNOS Gene Therapy in Established Disease

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance caused by narrowing and loss of pulmonary microvasculature, which in its late stages becomes refractory to traditional therapies. We hypothesized that bone marrow–derived endothelial progenitor cells (EPCs), which normally function to repair and regenerate blood vessels, would restore pulmonary hemodynamics and increase microvascular perfusion in the rat monocrotaline (MCT) model of PAH. Mononuclear cells were isolated from the bone marrow of syngeneic Fisher-344 rats by Ficoll gradient centrifugation and cultured for 7 to 10 days in endothelial growth medium. Fluorescently labeled endothelial-like progenitor cells (ELPCs) engrafted at the level of the distal pulmonary arterioles and incorporated into the endothelial lining in the MCT-injured lung. The administration of ELPCs 3 days after MCT nearly completely prevented the increase in right ventricular systolic pressure seen at 3 weeks with MCT alone (31.5±0.95 versus 48±3 mm Hg, respectively; P<0.001), whereas injection of skin fibroblasts had no protective effect (50.9±5.4 mm Hg). Delayed administration of progenitor cells 3 weeks after MCT prevented the further progression of PAH 2 weeks later (ie, 5 weeks after MCT), whereas only animals receiving ELPCs transduced with human endothelial NO-synthase (eNOS) exhibited significant reversal of established disease at day 35 (31±2 mm Hg, P<0.005) compared with day 21 (50±3 mm Hg). Fluorescent microangiography revealed widespread occlusion of pulmonary precapillary arterioles 3 weeks after MCT, whereas arteriolar-capillary continuity and microvascular architecture was preserved with the administration of syngeneic ELPCs. Moreover, the delivery of ELPCs to rats with established PAH resulted in marked improvement in survival, which was greatest in the group receiving eNOS-transduced cells. We conclude that bone marrow–derived ELPCs can engraft and repair the MCT-damaged lung, restoring microvasculature structure and function. Therefore, the regeneration of lung vascular endothelium by injection of progenitor cells may represent a novel treatment paradigm for patients with PAH.

Abnormal Aortic Valve Development in Mice Lacking Endothelial Nitric Oxide Synthase

BACKGROUND Endothelium-derived nitric oxide (NO) is produced by an oxidative reaction catalyzed by endothelial NO synthase (eNOS). NO plays a crucial role in controlling cell growth and apoptosis, as well as having well-characterized vasodilator and antithrombotic actions. More recently, endothelium-derived NO was shown to be involved in postdevelopmental vascular remodeling and angiogenesis, as well as in the formation of limb vasculature during embryogenesis. Therefore, we investigated the role of endothelium-derived NO during cardiovascular development using mice deficient in eNOS. METHODS AND RESULTS We examined the hearts of 12 mature eNOS-deficient and 26 mature wild-type mice. Five of the mature eNOS-deficient mice had a bicuspid aortic valve; none of the 26 wild-type animals exhibited identifiable valvular or cardiac abnormalities. Immunohistochemical analysis revealed prominent eNOS expression localized to the endothelium lining the valve cusps of the aorta in mature wild-type mice; expression was localized to the myocardium and endothelial cell monolayer lining the valve leaflets in the developing embryo. CONCLUSIONS These results show a strong association between eNOS deficiency and the presence of a bicuspid aortic valve; they provide the first molecular insight into one of the most common types of congenital cardiac abnormality.

Bone Morphogenetic Protein Receptor-2 Signaling Promotes Pulmonary Arterial Endothelial Cell Survival Implications for Loss-of-Function Mutations in the Pathogenesis of Pulmonary Hypertension

Mutations in the bone morphogenetic protein (BMP) receptor-2 (BMPR2) have been found in patients with idiopathic pulmonary arterial hypertension (IPAH); however, the mechanistic link between loss of BMPR2 signaling and the development of pulmonary arterial hypertension is unclear. We hypothesized that, contrary to smooth muscle cells, this pathway promotes survival in pulmonary artery endothelial cells (ECs) and loss of BMPR2 signaling will predispose to EC apoptosis. ECs were treated with BMP-2 or BMP-7 (200 ng/mL) for 24 hours in regular or serum-free (SF) medium, with and without addition of tumor necrosis factor &agr;, and apoptosis was assessed by flow cytometry (Annexin V), TUNEL, or caspase-3 activity. Treatment for 24 hours in SF medium increased apoptosis, and both BMP-2 and BMP-7 significantly reduced apoptosis in response to serum deprivation to levels not different from serum controls. Transfection with 5 &mgr;g of small interfering RNAs for BMPR2 produced specific gene silencing assessed by RT-PCR and Western blot analysis. BMPR2 gene silencing increased apoptosis almost 3-fold (P=0.0027), even in the presence of serum. Circulating endothelial progenitor cells (EPCs) isolated from normal subjects or patients with IPAH were differentiated in culture for 7 days and apoptosis was determined in the presence and absence of BMPs. BMP-2 reduced apoptosis induced by serum withdrawal in EPCs from normal subjects but not in EPCs isolated from patients with IPAH. These results support the hypothesis that loss-of-function mutations in BMPR2 could lead to increased pulmonary EC apoptosis, representing a possible initiating mechanism in the pathogenesis of pulmonary arterial hypertension.

Acellular Matrix: A Biomaterials Approach for Coronary Artery Bypass and Heart Valve Replacement

We have developed a multistep detergent-enzymatic extraction process (involving hypotonic and hypertonic solutions, the detergents octyl-phenoxy-polyethoxyethanol and sodium dodecyl sulfate, as well as DNAse and RNAse) which, while inhibiting autolysis, removes all cells from tissues and, with them, cellular antigens together with lipids and more soluble glycosaminoglycans. What remains is acellular matrix with the structural proteins well conserved and normally arranged. Canine arteries extracted to acellular matrix were implanted as coronary artery bypass allografts in a canine model, without the use of cardiopulmonary bypass, and compared with autogenous saphenous veins. Of nine pilot acellular matrix implants, four were patent, as compared with four of seven saphenous vein grafts. All occlusions in both graft types occurred acutely soon after implantation, with almost all patent grafts followed up for 6 months. The acellular matrix allografts showed no inflammation and only minimal cellular repopulation. This model needs further development, but appears promising for preclinical evaluation. Canine aortic and pulmonic valves extracted to acellular matrix using a modification of our extraction process, eliminating the detergent sodium dodecyl sulfate, were implanted heterotopically as allografts in the left main pulmonary artery in dogs, a location chosen to avoid the need for cardiopulmonary bypass. At 1 month, two-dimensional echocardiography of six implants showed leaflet motion and 3- to 5-mm Hg transvalvular gradients. Explant histology of four valves at 1 month showed no inflammation, cellular repopulation at the base of the valve, and partial endothelialization.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis (Programmed Cell Death) in Arteries of the Neonatal Lamb

We have examined whether cell death contributes to postnatal remodeling of arteries in lambs. First, abdominal aortic smooth muscle cell proliferation rates fell from 2.87 +/- 0.08% per day at 3 days of age to 1.75 +/- 0.15% per day at 21 days. These proliferation rates would yield a 50% increase in DNA content in the absence of cell death. No increase in DNA content was observed (P < .05 for predicted versus measured accumulation); therefore, significant cell death was inferred. The same analysis did not indicate high cell-death rates in the carotid, renal, or iliac arteries; however, cell death was detected in situ by end-labeling partially degraded DNA with terminal deoxynucleotidyl transferase or by nuclear labeling with propidium iodide, a fluorescent dye that permeates only nonviable cells. Nuclei were labeled in all arteries, although labeling was most frequent in the abdominal aorta, a vessel that regresses substantially after birth. Cell death was apoptotic because DNA extracted from arteries and end-labeled with [32P]dCTP produced a series of low molecular weight bands (DNA ladder) on an agarose gel, a hallmark of apoptosis. The ladder was strong for neonatal abdominal aorta but weak for other arteries. Only weak laddering was observed for fetal abdominal aortas in late gestation, confirming that high apoptosis rates in this vessel were initiated after birth. Intense DNA ladders and frequent in situ labeling indicated high rates of apoptosis in the postnatal intra-abdominal umbilical artery, another vessel that regresses after birth. We conclude that apoptosis contributes to postpartum arterial remodeling. This contribution is greatest in arteries that regress after birth.

The role of crosslinking in modification of the immune response elicited against xenogenic vascular acellular matrices

We have used detergent and enzymatic extraction of natural arteries to produce an acellular matrix vascular prosthesis (AMVP). Implanted as an allograft in a canine model, this AMVP shows excellent handling characteristics, low thromboreactivity, no evidence of aneurysm, and exceptional graft patency in the peripheral vasculature. As a first step in the development of xenograft AMVPs, we processed caprine carotid arteries to AMVP and implanted them as femoral interposition grafts in dogs. Explanted xenografts at 4 weeks showed multifocal mixed inflammatory infiltrates and focal destruction of the medial elastin in the inflammatory foci. To further study the immune response to xenogenic AMVP, we implanted canine-derived AMVPs and fresh canine arteries for 4 weeks in a Lewis rat model. Extraction to AMVP markedly reduced the circulating antibody response to the xenogenic implants; however, histological analysis revealed that both xenograft arteries and AMVPs produced a marked immune response with penetration of mononuclear cells into the media and adventitia. To modify the immune response, we applied three crosslinking techniques to the canine AMVPs: glutaraldehyde, polyglycidyl ether, and carbodiimide. All crosslinkers significantly reduced degradation and cellular infiltration of the prostheses. However, crosslinking neither eliminated the chronic inflammatory response surrounding the implants nor reduced the humoral response to the xenogenic materials.

Single-cell hydrogel encapsulation for enhanced survival of human marrow stromal cells

Inadequate extracellular matrix cues and subsequent apoptotic cell death are among crucial factors currently limiting cell viability and organ retention in cell-based therapeutic strategies for vascular regeneration. Here we describe the use of a single-cell hydrogel capsule to provide enhanced cell survival of adherent cells in transient suspension culture. Human marrow stromal cells (hMSCs) were singularly encapsulated in agarose capsules containing the immobilized matrix molecules, fibronectin and fibrinogen to ameliorate cell-matrix survival signals. MSCs in the enriched capsules demonstrated increased viability, greater metabolic activity and enhanced cell-cytoskeletal patterning. Increased cell viability resulted from the re-induction of cell-matrix interactions likely via integrin clustering and subsequent activation of the extracellular signal regulated MAPK (ERK)/mitogen activated protein kinase (MAPK) signaling cascade. Proof of principle in-vivo studies, investigating autologous MSC delivery into Fisher 344 rat hindlimb, depicted a significant increase in the number of engrafted cells using the single-cell encapsulation system. Incorporation of immobilized adhesion molecules compensates, at least in part, for the missing cell-matrix cues, thereby attenuating the initial anoikis stimuli and providing protection from subsequent apoptosis. Thus, this single-cell encapsulation strategy may markedly enhance therapeutic cell survival in targeted tissues.

Innate Immunity in the Therapeutic Actions of Endothelial Progenitor Cells in Pulmonary Hypertension

Direct injection of endothelial progenitor cells (EPCs) into the circulation has shown therapeutic benefit in both experimental models and clinical studies of pulmonary arterial hypertension (PAH). Using the monocrotaline (MCT)-induced rat model of PAH, we investigated the role of innate immunity in the therapeutic activity of two types of putative EPCs derived from human peripheral blood mononuclear cells: an early population of endothelial-like, culture-modified monocytes (E-CMMs) and late-outgrowth EPCs (L-EPCs), which exhibit a strong endothelial phenotype. In the athymic nude rat, E-CMMs prevented MCT-induced increases in right ventricular systolic pressure (P < 0.001) and right ventricular hypertrophy (P < 0.01) when administered 3 days after MCT challenge, whereas L-EPCs were ineffective. However, in both cases, there was a lack of cell persistence within the lungs at 24 hours after injection, likely due to residual natural killer (NK) cell activity in the model. Although ablation of NK and NK-T cells with anti-asialo-GM-1 antiserum enhanced the retention of both E-CMMs and L-EPCs, still no benefit was seen with L-EPCs, and the efficacy of E-CMMs was lost. In vitro characterization revealed that E-CMMs resemble a regulatory subtype of dendritic cells, producing IL-10, but not IL-12, in response to inflammatory stimuli. Coculture studies demonstrated the capacity of E-EPCs to stimulate autologous human and nude rat NK cells in vitro. These data support a novel mode of action for human E-CMMs in the prevention of PAH, whereby they act through an immune-dependent mechanism, potentially involving the stimulation of NK cells.

Capture of flowing endothelial cells using surface-immobilized anti-kinase insert domain receptor antibody.

In humans, self-endothelialization of synthetic grafts is severely limited, but a recent interesting idea is to attract endothelial progenitor cells (EPCs) from peripheral blood onto grafts via antibodies directed at proposed EPC markers. Results with anti-CD34 antibodies have shown some promise, but it is unclear whether CD34 is the best marker for cells with re-endothelializing potential. Much evidence points to kinase insert domain receptor (KDR) as an important indicator of endothelial potential if not a definitive marker. Because KDR is not an adhesion molecule (like CD34), we first demonstrated the ability to use adsorbed and protein G-oriented antibody to this receptor to capture flowing cells onto a solid surface. Using endothelial cells and smooth muscle cells, we show in a model system under low shear rates the ability to selectively capture cells by this receptor. Furthermore, our results indicate that concomitant flow of cells lacking the receptor does not affect the efficiency of capture of KDR(+) cells but that orienting the antibody significantly increases the efficiency of capture.

A nonantibiotic chemically modified tetracycline (CMT-3) inhibits intimal thickening.

Recent research has shown that the tetracycline antibiotics are pluripotent drugs that inhibit the activity of matrix metalloproteinases (MMPs) and affect many cellular functions including proliferation, migration, and matrix remodeling. We have shown that doxycycline inhibits MMP activity and intimal thickening after injury of the rat carotid artery, however we do not know whether these effects are because of the antibiotic, anti-MMP, or other actions of doxycycline. Recently, chemically modified tetracyclines have been synthesized that lack antibiotic activity but retain anti-MMP activity (CMT-3), or lack both antibiotic and anti-MMP activity (CMT-5). In the current study we have assessed the effects of treatment with CMT-3 or CMT-5 on intimal thickening after balloon catheter injury of the rat carotid artery. Rats were treated by oral gavage with 15 mg/kg/day CMT-3 or CMT-5. CMT-3 significantly reduced smooth muscle cell (SMC) proliferation in both the medial and intimal layers of the injured rat carotid artery compared to CMT-5. Furthermore, CMT-3 inhibited SMC migration from the media to the intima by 86% at 4 days after injury. CMT-3 also decreased MMP-2 activity. Finally, we found that CMT-3 treatment resulted in a significant reduction in intimal cross-sectional area from 0.23 +/- 0.01 mm(2) in the CMT-5 control group to 0.19 +/- 0.01 mm(2). There was also a reduction in elastin and collagen accumulation within the intima. We conclude that CMT-3 attenuated intimal thickening after arterial injury by inhibiting SMC proliferation, migration and MMP activity, and accumulation of extracellular matrix. The inhibitory effects of CMT-3 were independent of the antibiotic properties, but were dependent on the anti-MMP activity of the tetracycline family.