Targeting extracellular vesicle delivery to the lungs by microgel encapsulation.

Extracellular vesicles (EVs) secreted by stem and progenitor cells have significant potential as cell-free 'cellular' therapeutics. Yet, small EVs (<200 nm) are rapidly cleared after systemic administration, mainly by the liver, presenting challenges targeting EVs to a specific organ or tissue. Microencapsulation using natural nano-porous hydrogels (microgels) has been shown to enhance engraftment and increase the survival of transplanted cells. We sought to encapsulate EVs within microgels to target their delivery to the lung by virtue of their size-based retention within the pulmonary microcirculation. Mesenchymal stromal cell (MSC) derived EVs were labelled with the lipophilic dye (DiR) and encapsulated within agarose-gelatin microgels. Endothelial cells and bone marrow derived macrophages were able to take up EVs encapsulated in microgels in vitro, but less efficiently than the uptake of free EVs. Following intrajugular administration, microgel encapsulated EVs were selectively retained within the lungs for 72h, while free EVs were rapidly cleared by the liver. Furthermore, microgel-loaded EVs demonstrated greater uptake by lung cells, in particular CD45+ immune cells, as assessed by flow cytometry compared to free EVs. Microencapsulation of EVs may be a novel tool for enhancing the targeted delivery of EVs for future therapeutic applications.

Mesenchymal stem cell therapy and cognition in MS: Preliminary findings from a phase II clinical trial.

BACKGROUND: Mesenchymal stem cell (MSC) therapies are being evaluated in multiple sclerosis (MS) for possible neural repair. To date, the potential benefits on cognition have received little attention. The objective of the current study was to comprehensively evaluate cognition before and after MSC therapy in those with MS as part of a double-blind, phase II clinical trial. METHODS: Twenty-eight individuals with a confirmed diagnosis of MS were randomly assigned into two study arms. Cognition was evaluated using an expanded Minimal Assessment of Cognitive Function in Multiple Sclerosis (MACFIMS) battery. The battery was administered at Week 0, Week 24, and Week 48 and results were analysed at the group and individual level. RESULTS: No detectable effect of MSC-mediated neural repair was noted in the short-term with respect to cognition, although some cognitive stability or improvement was observed. Decline was noted in some cognitive areas immediately following the procedure at Week 24; though these were temporary with performance returning to baseline levels at Week 48. CONCLUSIONS: While MSC therapy does not lead to improvement in cognition, at least in the short-term, neither does the procedure have lasting deleterious effects. The current findings lend support to the safety and feasibility of MSC therapy as a potentially viable treatment option for individuals with MS.

Engineering blood outgrowth endothelial cells to optimize endothelial nitric oxide synthase and extracellular matrix production for coating of blood contacting surfaces.

Coverage of blood contacting surfaces by a functional endothelial layer is likely required to induce and maintain homeostasis. Blood outgrowth endothelial cells (BOECs), cultured from human peripheral blood monocytes, are readily available and functional autologous endothelial source that may represent a reasonable alternative to vascular derived cells. Endothelial nitric oxide synthase (eNOS) produces NO, an important factor that regulates homeostasis at the blood-contacting surface. We found that BOECs express markedly lower levels of eNOS protein (34% ± 13%, Western blot) and mRNA (29% ± 17%, qRT-PCR), as well as exhibiting reduced activity (49% ± 18%, Nitrite analysis) when compared to human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells. HUVECs grown on fibronectin, type I collagen, or laminin -coated surfaces exhibited significant reduction of eNOS mRNA and protein expression. However, no decrease in eNOS levels was observed in BOECs. Interestingly BOECs expressed significantly higher Collagen (Col) I compared to HUVECs, and blocking Col I synthesis significantly enhanced eNOS expression in BOECs. Inhibition of ß1 integrin, focal adhesion kinase (FAK), or actin polymerization increased eNOS in both BOECs and HUVECs suggesting involvement of a signaling pathway culminating in stabilization of the cytoskeleton. Finally, we demonstrated that a Rho-associated protein kinases (ROCK) inhibitor, as a disruptor of actin stabilization, enhanced both eNOS expression and bioactivity. Taken together, our findings demonstrate that cell-ECM interactions are fundamental to the regulation of eNOS in BOECs and suggest that disruption of key intracellular pathways (such as ROCK) may be necessary to enhance functional activity of an endothelialized surface. STATEMENT OF SIGNIFICANCE: Development of biocompatible blood-contacting biomaterial surfaces has not been possible to date, leading many investigators to believe that a complete autologous endothelial layer will be necessary. Blood outgrowth endothelial cells (BOECs), cultured from human peripheral blood monocytes, are readily available and functional autologous endothelial source. Endothelial nitric oxide synthase (eNOS) produces NO, an important factor that regulates homeostasis at the blood-contacting surface. In this study, we show that eNOS displays limited expression in cultured BOECs. We further demonstrate that a strong negative regulation of eNOS is mediated by collagen substrates and that treatment with ROCK inhibitor could enhance both eNOS expression and activity in BOECs and help to rapidly establish a functional autologous endothelial layer on cardiovascular biomaterials.

Differentiation of Murine Bone Marrow-Derived Smooth Muscle Progenitor Cells Is Regulated by PDGF-BB and Collagen.

Smooth muscle cells (SMCs) are key regulators of vascular disease and circulating smooth muscle progenitor cells may play important roles in vascular repair or remodelling. We developed enhanced protocols to derive smooth muscle progenitors from murine bone marrow and tested whether factors that are increased in atherosclerotic plaques, namely platelet-derived growth factor-BB (PDGF-BB) and monomeric collagen, can influence the smooth muscle specific differentiation, proliferation, and survival of mouse bone marrow-derived progenitor cells. During a 21 day period of culture, bone marrow cells underwent a marked increase in expression of the SMC markers α-SMA (1.93 ± 0.15 vs. 0.0008 ± 0.0003 (ng/ng GAPDH) at 0 d), SM22-α (1.50 ± 0.27 vs. 0.005 ± 0.001 (ng/ng GAPDH) at 0 d) and SM-MHC (0.017 ± 0.004 vs. 0.001 ± 0.001 (ng/ng GAPDH) at 0 d). Bromodeoxyuridine (BrdU) incorporation experiments showed that in early culture, the smooth muscle progenitor subpopulation could be identified by high proliferative rates prior to the expression of smooth muscle specific markers. Culture of fresh bone marrow or smooth muscle progenitor cells with PDGF-BB suppressed the expression of α-SMA and SM22-α, in a rapidly reversible manner requiring PDGF receptor kinase activity. Progenitors cultured on polymerized collagen gels demonstrated expression of SMC markers, rates of proliferation and apoptosis similar to that of cells on tissue culture plastic; in contrast, cells grown on monomeric collagen gels displayed lower SMC marker expression, lower growth rates (319 ± 36 vs. 635 ± 97 cells/mm2), and increased apoptosis (5.3 ± 1.6% vs. 1.0 ± 0.5% (Annexin 5 staining)). Our data shows that the differentiation and survival of smooth muscle progenitors are critically affected by PDGF-BB and as well as the substrate collagen structure.

Marked Strain-Specific Differences in the SU5416 Rat Model of Severe Pulmonary Arterial Hypertension.

We assessed the pulmonary hemodynamic response to vascular endothelial growth factor receptor, type 2, inhibition using SU5416 (SU) with and without chronic hypoxia (CH) in different background strains and colonies of rats. A single subcutaneous injection of SU (20 mg/kg) or vehicle was administered to different substrains of Sprague-Dawley (SD) rats, and they were compared with Lewis and Fischer rats, with and without exposure to CH (10% O2 for 3 wk). Remarkably, a unique colony of SD rats from Charles River Laboratories, termed the SD-hyperresponsive type, exhibited severe pulmonary arterial hypertension (PAH) with SU alone, characterized by increased right ventricular systolic pressure, right ventricular/left ventricular plus septal weight ratio, and arteriolar occlusive lesions at 7-8 weeks (all P < 0.0001 versus vehicle). In contrast, the other SD substrain from Harlan Laboratories, termed SD-typical type, as well as Fischer rats, developed severe PAH only when exposed to SU and CH, whereas Lewis rats showed only a minimal response. All SD-typical type rats survived for up to 13 weeks after SU/CH, whereas SD-hyperresponsive type rats exhibited mortality after SU and SU/CH (35% and 50%, respectively) at 8 weeks. Fischer rats exposed to SU/CH exhibited the greatest mortality at 8 weeks (78%), beginning as early as 4 weeks after SU and preceded by right ventricle enlargement. Of note, a partial recovery of PAH after 8 weeks was observed in the SD-typical type substrain only. In conclusion, variation in strain, even between colonies of the same strain, has a remarkable influence on the nature and severity of the response to SU, consistent with an important role for genetic modifiers of the PAH phenotype.

Endothelial NO-Synthase Gene-Enhanced Progenitor Cell Therapy for Pulmonary Arterial Hypertension: The PHACeT Trial.

RATIONALE: Pulmonary arterial hypertension (PAH) remains a progressive and eventually lethal disease characterized by increased pulmonary vascular resistance because of loss of functional lung microvasculature, primarily at the distal (intracinar) arteriolar level. Cell-based therapies offer the potential to repair and regenerate the lung microcirculation and have shown promise in preclinical evaluation in experimental models of PAH. OBJECTIVE: The Pulmonary Hypertension and Angiogenic Cell Therapy (PHACeT) trial was a phase 1, dose-escalating clinical study of the tolerability of culture-derived endothelial progenitor cells, transiently transfected with endothelial nitric oxide synthase, in patients with PAH refractory to PAH-specific therapies. METHODS AND RESULTS: Seven to 50 million endothelial nitric oxide synthase-transfected endothelial progenitor cells, divided into 3 doses on consecutive days, were delivered into the right atrium via a multiport pulmonary artery catheter during continuous hemodynamic monitoring in an intensive care unit setting. Seven patients (5 women) received treatment from December 2006 to March 2010. Cell infusion was well tolerated, with no evidence of short-term hemodynamic deterioration; rather, there was a trend toward improvement in total pulmonary resistance during the 3-day delivery period. However, there was 1 serious adverse event (death) which occurred immediately after discharge in a patient with severe, end stage disease. Although there were no sustained hemodynamic improvements at 3 months, 6-minute walk distance was significantly increased at 1, 3, and 6 months. CONCLUSION: Delivery of endothelial progenitor cells overexpressing endothelial nitric oxide synthase was tolerated hemodynamically in patients with PAH. Furthermore, there was evidence of short-term hemodynamic improvement, associated with long-term benefits in functional and quality of life assessments. However, future studies are needed to further establish the efficacy of this therapy. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00469027.

Proteomic analysis implicates translationally controlled tumor protein as a novel mediator of occlusive vascular remodeling in pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular endothelial cells (ECs). Hereditary PAH (HPAH) is often caused by mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2). However, the mechanisms by which these mutations cause PAH remain unclear. Therefore, we screened for dysregulated proteins in blood-outgrowth ECs of HPAH patients with BMPR2 mutations compared with healthy control subjects. METHODS AND RESULTS: A total of 416 proteins were detected with 2-dimensional PAGE in combination with liquid chromatography/tandem mass spectrometry analysis, of which 22 exhibited significantly altered abundance in blood-outgrowth ECs from patients with HPAH. One of these proteins, translationally controlled tumor protein (TCTP), was selected for further study because of its well-established role in promoting tumor cell growth and survival. Immunostaining showed marked upregulation of TCTP in lungs from patients with HPAH and idiopathic PAH, associated with remodeled vessels of complex lesions. Increased TCTP expression was also evident in the SU5416 rat model of severe and irreversible PAH, associated with intimal lesions, colocalizing with proliferating ECs and the adventitia of remodeled vessels but not in the vascular media. Furthermore, silencing of TCTP expression increased apoptosis and abrogated the hyperproliferative phenotype of blood-outgrowth ECs from patients with HPAH, raising the possibility that TCTP may be a link in the emergence of apoptosis-resistant, hyperproliferative vascular cells after EC apoptosis. CONCLUSION: Proteomic screening identified TCTP as a novel mediator of endothelial prosurvival and growth signaling in PAH, possibly contributing to occlusive pulmonary vascular remodeling triggered by EC apoptosis.

The effect of encapsulation of cardiac stem cells within matrix-enriched hydrogel capsules on cell survival, post-ischemic cell retention and cardiac function.

Transplantation of ex vivo proliferated cardiac stem cells (CSCs) is an emerging therapy for ischemic cardiomyopathy but outcomes are limited by modest engraftment and poor long-term survival. As such, we explored the effect of single cell microencapsulation to increase CSC engraftment and survival after myocardial injection. Transcript and protein profiling of human atrial appendage sourced CSCs revealed strong expression the pro-survival integrin dimers αVß3 and α5ß1- thus rationalizing the integration of fibronectin and fibrinogen into a supportive intra-capsular matrix. Encapsulation maintained CSC viability under hypoxic stress conditions and, when compared to standard suspended CSC, media conditioned by encapsulated CSCs demonstrated superior production of pro-angiogenic/cardioprotective cytokines, angiogenesis and recruitment of circulating angiogenic cells. Intra-myocardial injection of encapsulated CSCs after experimental myocardial infarction favorably affected long-term retention of CSCs, cardiac structure and function. Single cell encapsulation prevents detachment induced cell death while boosting the mechanical retention of CSCs to enhance repair of damaged myocardium.

Derivation of human peripheral blood derived endothelial progenitor cells and the role of osteopontin surface modification and eNOS transfection.

Endothelial coverage of blood-contacting biomaterial surfaces has been difficult to achieve. A readily available autologous source of endothelium combined with an appropriate attachment substrate would improve the chances of developing functional surfaces. Here we describe methods to derive high quantities of human endothelial progenitor cells (EPCs) from peripheral blood monocytes (PBMCs) obtained by leukapheresis. These cells are morphologically and phenotypically similar to human umbilical vein endothelial cells (HUVECs); however, their expression of the key vascular factor - endothelial nitric oxide synthase (eNOS) - is markedly lower than that observed in HUVECs. We demonstrate that eNOS levels can be restored with plasmid-based transfection. To promote EPC adherence we examined substrate enhancement with a matricellular protein associated with vascular repair, osteopontin (OPN). We observed dose- and time-dependent responses of OPN in EPC adhesion, spreading, and haptotactic migration of EPCs in Boyden chamber assays. In addition, the combination of the OPN coating and enhanced eNOS expression in EPCs maximally enhanced cell adhesion (39.6 ± 1.7 and 49.4 ± 2.4 cells/field for 0 and 1 nM OPN) and spreading (84.7 ± 3.5% and 92.1 ± 3.9% for 0 nM and 1 nM OPN). These data highlight the direct effects of OPN on peripheral blood derived EPCs, suggesting that OPN works by mediating progenitor cell adhesion during vascular injury. The combination of autologous EPCs and OPN coatings could be a promising method of developing functional endothelialized surfaces.

A lymphocyte-dependent mode of action for imatinib mesylate in experimental pulmonary hypertension.

The capacity of imatinib mesylate to reverse established pulmonary arterial hypertension (PAH) has been attributed to a reduction in pulmonary arterial muscularization via inhibition of platelet-derived growth factor receptor-ß on vascular smooth muscle cells. However, there is also a significant immunomodulatory component to the action of imatinib that may account for its efficacy in PAH. We found that monocrotaline-induced pulmonary hypertension was associated with a significant decrease in pulmonary natural killer (NK) cells and T lymphocytes and the accumulation of macrophages in the lungs of F344 rats. The prevention of pulmonary hypertension by imatinib blocked these changes in pulmonary leukocyte content and induced elevations in pulmonary interferon-γ, tumor necrosis factor α, and IL-10, corresponding to the enhanced activity of splenic NK cells ex vivo. Treatment with anti-asialo GM1 antiserum (ASGM1), which ablated circulating NK cells and depleted T cells, eliminated the therapeutic benefit of imatinib. ASGM1-treated animals also exhibited significant pulmonary arteriolar muscularization in response to monocrotaline challenge compared with immunocompetent controls despite daily imatinib administration to both groups. In the athymic rat, imatinib decreased right ventricular hypertrophy and pulmonary arteriolar muscularization in monocrotaline-challenged animals versus saline-treated controls but did not prevent pulmonary macrophage accumulation or the development of pulmonary hypertension. These data demonstrate that the immunomodulatory effects of imatinib are critical to its therapeutic action in experimental PAH.

Differential roles of endothelin-1 in angiotensin II-induced atherosclerosis and aortic aneurysms in apolipoprotein E-null mice.

Because both endothelin-1 (ET-1) and angiotensin II (AngII) are independent mediators of arterial remodeling, we sought to determine the role of ET receptor inhibition in AngII-accelerated atherosclerosis and aortic aneurysm formation. We administered saline or AngII and/or bosentan, an endothelin receptor antagonist (ERA) for 7, 14, or 28 days to 6-week- and 6-month-old apolipoprotein E-knockout mice. AngII treatment increased aortic atherosclerosis, which was reduced by ERA. ET-1 immunostaining was localized to macrophage-rich regions in aneurysmal vessels. ERA did not prevent AngII-induced aneurysm formation but instead may have increased aneurysm incidence. In AngII-treated animals with aneurysms, ERA had a profound effect on the non-aneurysmal thoracic aorta via increasing wall thickness, collagen/elastin ratio, wall stiffness, and viscous responses. These observations were confirmed in acute in vitro collagen sheet production models in which ERA inhibited AngII's dose-dependent effect on collagen type 1 α 1 (COL1A1) gene transcription. However, chronic treatment reduced matrix metalloproteinase 2 mRNA expression but enhanced COL3A1, tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2 mRNA expressions. These data confirm a role for the ET system in AngII-accelerated atherosclerosis but suggest that ERA therapy is not protective against the formation of AngII-induced aneurysms and can paradoxically stimulate a chronic arterial matrix remodeling response.

The role of transglutaminase 2 and osteopontin in matrix protein supplemented microencapsulation of marrow stromal cells.

Previous studies reported that matrix protein supplementation (fibronectin/fibrinogen, FN/FG) of agarose gel microcapsules enhances survival and target tissue retention of syngeneic rat marrow stromal cells (MSCs). We hypothesized that additional supplementation of microcapsules with osteopontin (OPN) and transglutaminase 2 (TG2) would enhance cell survival, while stabilizing the provisional matrix. Using monomeric OPN or OPN polymerized with TG2, we examined human MSC adhesion, morphology, focal contact formation and apoptosis. Polymeric OPN with TG2 induced greater adhesion than monomeric OPN (84.5 ± 10.7 vs. 44.3 ± 10.0 cells/field), and also significantly enhanced focal contact formation (351.5 ± 21.2 vs. 45.6 ± 17.6 focal contact sites/cell) and cell spreading (2.7 × 10(3) ± 0.20 × 10(3) µm(2) vs. 1.2 × 10(3) ± 0.26 × 10(2) µm(2)) while preserving MSC pluripotency. Microcapsules supplemented with FN/FG, polymeric OPN and TG2 demonstrated significantly less apoptotic cells than FN/FG microcapsules (14.0 ± 2.34% vs. 28.2 ± 3.22%). Reduced apoptosis was attributed to matrix stabilization by TG2 and the synergistic activity of matrix proteins. It is anticipated that this enhanced survival will maximize the therapeutic potential of MSCs.

Rationale and design of Enhanced Angiogenic Cell Therapy in Acute Myocardial Infarction (ENACT-AMI): the first randomized placebo-controlled trial of enhanced progenitor cell therapy for acute myocardial infarction.

BACKGROUND: Despite the widespread use of pharmacological and/or interventional reperfusion therapies, recovery of cardiac function in myocardial infarction (MI) patients is often modest or even absent. Unlike classical pharmacological treatments, the use of progenitor cells could potentially restore functional tissue in regions that otherwise would form only scar. However, a major limitation of autologous cell therapy is the deleterious influence of age and cardiac risk factors on progenitor cell activity. TRIAL DESIGN: The ENACT-AMI trial is a phase IIb, double-blind, randomized placebo-controlled trial, using transplantation of autologous early endothelial progenitor cells (EPCs) for patients who have suffered large MI. Circulating mononuclear cells (MNCs) are obtained by apheresis and subjected to differential culture for 3 days to select a population of highly regenerative, endothelial-like, culture modified MNCs (E-CMMs), often referred to as "early EPCs." A total of 99 patients will be randomized to placebo (Plasma-Lyte A), autologous E-CMMs, or E-CMMs transfected with human endothelial nitric oxide synthase delivered by coronary injection into the infarct-related artery. The primary efficacy end point is change from baseline to 6 months in global left ventricular ejection fraction by cardiac MRI; secondary endpoints include regional wall motion, wall thickening, infarct volume, time to clinical worsening, and quality of life. CONCLUSIONS: This will be the first clinical trial to include a strategy designed to enhance the function of autologous progenitor cells by overexpressing endothelial nitric oxide synthase, and the first to use combination gene and cell therapy for the treatment of cardiac disease.

Enhanced translation of heme oxygenase-2 preserves human endothelial cell viability during hypoxia.

Heme oxygenases (HOs) -1 and -2 catalyze the breakdown of heme to release carbon monoxide, biliverdin, and ferrous iron, which may preserve cell function during oxidative stress. HO-1 levels decrease in endothelial cells exposed to hypoxia, whereas the effect of hypoxia on HO-2 expression is unknown. The current study was carried out to determine if hypoxia alters HO-2 protein levels in human endothelial cells and whether this enzyme plays a role in preserving their viability during hypoxic stress. Human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), and human blood outgrowth endothelial cells were exposed to 21% or 1% O(2) for 48 or 16 h in the presence or absence of tumor necrosis factor-alpha (10 ng/ml) or H(2)O(2) (100 microm). In all three endothelial cell types HO-1 mRNA and protein levels were decreased following hypoxic incubation, whereas HO-2 protein levels were unaltered. In HUVECs HO-2 levels were maintained during hypoxia despite a 57% reduction in steady-state HO-2 mRNA level and a 43% reduction in total protein synthesis. Polysome profiling revealed increased HO-2 transcript association with polysomes during hypoxia consistent with enhanced translation of these transcripts. Importantly, inhibition of HO-2 expression by small interference RNA increased oxidative stress, exacerbated mitochondrial membrane depolarization, and enhanced caspase activation and apoptotic cell death in cells incubated under hypoxic but not normoxic conditions. These data indicate that HO-2 is important in maintaining endothelial viability and may preserve local regulation of vascular tone, thrombosis, and inflammatory responses during reductions in systemic oxygen delivery.

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.

Elevated platelet angiostatin and circulating endothelial microfragments in idiopathic pulmonary arterial hypertension: a preliminary study.

INTRODUCTION: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by pulmonary arteriolar narrowing and degeneration associated with in situ thrombosis. We hypothesized that microvascular endothelial injury and apoptosis may be an initiating mechanism in IPAH. Endothelial apoptosis generates endothelial microfragments (EMF), which can activate platelets. Platelets release both VEGF and angiostatin, which depending the balance can inhibit or induce endothelial apoptosis, respectively. MATERIALS AND METHODS: We measured EMFs from blood of IPAH patients as index of endothelial cell apoptosis/injury and levels of pro- and anti- EC apoptotic factors found in platelets. EMFs and platelets in blood samples from control subjects and patients with IPAH were measured using a 4-color flow cytometry protocol, and platelet levels of VEGF and angiostatin were determined by ELISAs and immunoblotting. RESULTS: Compared to controls, IPAH patients exhibited higher numbers of circulating EMFs and more activated/apoptotic platelets. IPAH patients also exhibited higher levels of platelet angiostatin; however, no significant difference was detected in platelet VEGF levels between the two groups. CONCLUSIONS: These results are consistent with an increase in EC dysfunction in patients with IPAH, possibly contributing to the progression of IPAH and its associated thrombosis.

The enzymatic degradation of hyaluronan is associated with disease progression in experimental pulmonary hypertension.

Hyaluronan (HA) degradation fragments have been linked to inflammation in a wide range of lung diseases. In idiopathic pulmonary arterial hypertension, HA accumulation has been associated with advanced disease. In this study, we investigated the potential role of HA degradation in the early stages of disease by examining HA distribution, molecular mass, synthesis, and enzymatic degradation at different stages of disease progression in a rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH). At 28 days post-MCT, severe PH was associated with increased total lung HA (P = 0.04). In contrast, a significant decrease in total lung HA was observed on day 10, before the onset of PH (P = 0.02). Molecular mass analysis revealed a loss of high molecular mass (HMM) HA at 10 and 24 days post-MCT, followed by an increase in HMM HA at 28 days. Expression of HA synthase 2 (HAS2) was elevated in MCT-challenged animals at 24 and 28 days, consistent with increased synthesis of HMM HA. Analysis by Morgan Elson assay and zymography demonstrated increased hyaluronidase-1 activity in the lungs of MCT-challenged rats, indicating that the observed increases in HAS2 expression and HA synthesis were counterbalanced, in part, by enhanced degradation. The present data demonstrate that, in the MCT model, early-stage PH is associated with enhanced hyaluronidase-1 activity, while both degradation and synthesis are increased at later stages. Thus an early increase in the generation of proinflammatory HA fragments may play a role in the onset and progression of pulmonary arterial hypertension.

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.

A population analysis of VEGF transgene expression and secretion.

The induction of therapeutic angiogenesis with gene therapy approaches has received considerable interest and some limited clinical success. A major drawback to this approach is a lack of understanding of the pharmacokinetics of therapeutic protein delivery. This has become increasingly more relevant as recent studies have illustrated a defined therapeutic window for angiogenic protein secretion into the local microenvironment. For cell based gene therapies, with cells widely distributed throughout the tissue, this implies that any individual cell must attain a specific secretion rate to produce a local angiogenic response. Here we report a reproducible technique enabling the study of growth factor secretion from individual cells following transient plasmid transfection. We demonstrate significant variability in single cell vascular endothelial growth factor (VEGF) secretion with the majority of total protein secretion arising from a small subpopulation of transfected cells. We demonstrate that VEGF secretion is linearly correlated to intracellular plasmid copy number and protein secretion does not appear to reach saturation within the cell population. The selection of gene therapy approaches that optimize individual cell secretion profiles may be essential for the development of effective gene therapies.

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.