Search

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., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

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., (Copyright © 2022. Published by Elsevier B.V.)

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 β1 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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.

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.

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., (© 2015 American Heart Association, Inc.)

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., (© 2014 American Heart Association, Inc.)

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 αVβ3 and α5β1- 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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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-β 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., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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., (Copyright 2009 Elsevier Ltd. All rights reserved.)

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.

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.

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.

Agarose gel micro-droplets supplemented with provisional matrix proteins have been shown to enhance encapsulated cell survival for cell therapy applications. This study evaluated micro-droplet T(1) and T(2) relaxation on a 1.5 T clinical MRI scanner to guide the optimization of encapsulated cell delivery to intermediate-sized animals. Preliminary in vitro experiments using encapsulated human blood-derived endothelial progenitor cells (EPCs) documented a negligible impact of EPC encapsulation on agarose micro-droplet T(1) and T(2) relaxation, even following transient immersion in 2.3 mm Gd-DTPA. Furthermore, Gd-DTPA immersion did not adversely impact encapsulated cell viability. These results allowed for efficient pre-clinical methodological development using direct injections into the rabbit lumbar region of agarose droplets without cells (n=6). At time-points to 6 h, in vivo injection sites displayed elevated T(2) and T(1) (1.8%: DeltaT(2)=53+/-28%, DeltaT(1)=50+/-25%, n=13; 2.5%: DeltaT(2)=41+/-10%, DeltaT(1)=41+/-26%, n=11). Rapid imaging sequences displayed high conspicuity at sites of Gd-DTPA-immersed capsule injection, which persisted for less than 4 h. Therefore, basic differences of micro-droplet T(1) and T(2) when compared to tissue provide a platform for acute tracking of encapsulated cell fate. Transient Gd-DTPA encapsulation accentuates T(1) differences.

The purpose of this study was to determine if hypoxia elicits different proliferative and apoptotic responses in systemic arterial smooth muscle cells incubated under conditions that do or do not result in cellular ATP depletion and whether these effects are relevant to vascular remodeling in vivo. Gene expression profiling was used to identify potential regulatory pathways. In human aortic smooth muscle cells (HASMCs) incubated at 3% O(2), proliferation and progression through the G1/S interphase are enhanced. Incubation at 1% O(2) reduced proliferation, delayed G1/S transition, increased apoptotic cell death, and is associated with mitochondrial membrane depolarization and reduced cellular ATP levels. In aorta and mesenteric artery from rats exposed to hypoxia (10% O(2), 48 h), both proliferation and apoptosis are increased, as are medial nuclear density and smooth muscle cell content. Although nuclear levels of hypoxia-inducible factor 1-alpha (HIF-1alpha) are increased to a similar extent in HASMCs incubated at 1 and 3% O(2), expression of tumor protein p53, its transcriptional target p21, as well as their regulatory factors and downstream effectors, are differentially affected under these two conditions, suggesting that the bidirectional effects of hypoxia are mediated by this pathway. We conclude that hypoxia induces a state of enhanced cell turnover through increased rates of both smooth muscle cell proliferation and death. This confers the ability to remodel the vasculature in response to changing tissue metabolic needs while avoiding the accumulation of mutations that may lead to malignant transformation or the formation of abnormal vascular structures.

Pulmonary arterial hypertension (PAH) is characterized by widespread loss of pulmonary microvasculature. Therefore we hypothesized that angiogenic gene therapy would reverse established PAH, in part restoring the lung microcirculation. Three weeks after monocrotaline (MCT) treatment, Fisher 344 rats were randomized to receive a total of either 1.5 x 10(6) syngeneic fibroblasts (FB) transfected with vascular endothelial growth factor A (VEGF), endothelial NO synthase (eNOS), or null-plasmid transfected FBs. Right ventricular systolic pressure (RVSP) was similarly increased in all MCT-treated groups at the time of gene transfer. Animals receiving the null-vector progressed to severe PAH by Day 35 (P < 0.001). In contrast, eNOS gene transfer significantly reduced RVSP at Day 35 compared with Day 21, whereas VEGF prevented further increases in RVSP over the subsequent 2 wk but did not reverse established PAH. RV hypertrophy was significantly reduced in both the eNOS-treated and VEGF-treated groups compared with the null-transfected controls. Fluorescent microangiography revealed widespread occlusion of the pre-capillary arterioles 21 d after MCT treatment, and animals receiving eNOS gene transfer exhibited the greatest improvement in the arteriolar architecture and capillary perfusion at Day 35. Cell-based eNOS gene transfer was more effective than VEGF in reversing established PAH, associated with evidence of regeneration of pulmonary microcirculation.

Elastin, a principal structural component of native arteries, has distinct biological and mechanical advantages when used as a biomaterial; however, its low ultimate tensile strength has limited its use as an arterial conduit. We have developed a scaffold, consisting of a purified elastin tubular conduit strengthened with fibrin bonded layers of acellular small intestinal submucosa (aSIS) for potential use as a small diameter vascular graft. The addition of aSIS increased the ultimate tensile strength of the elastin conduits nine-fold. Burst pressures for the elastin composite vascular scaffold (1,396 +/- 309 mmHg) were significantly higher than pure elastin conduits (162 +/- 36 mmHg) and comparable to native saphenous veins. The average suture pullout strength of the elastin composite vascular scaffolds was 14.612 +/- 3.677 N, significantly higher than the pure elastin conduit (0.402 +/- 0.098 N), but comparable to native porcine carotid arteries (13.994 +/- 4.344 N). Cyclic circumferential strain testing indicated that the composite scaffolds were capable of withstanding physiological loading conditions for at least 83 h. Implantation of the elastin composites as carotid interposition grafts in swine demonstrated its superiority to clinically acceptable ePTFE with significantly longer average patency times of 5.23 h compared to 4.15 h. We have developed a biologically based elastin scaffold with suitable mechanical properties and low thrombogenicity for in vivo implantation, and with the potential for cellular repopulation and host integration reestablishing an appropriate elastic artery.

Visualization of the complex lung microvasculature and resolution of its three-dimensional architecture remains a difficult experimental challenge. We present a novel fluorescent microscopy technique to visualize both the normal and diseased pulmonary microvasculature. Physiologically relevant pulmonary perfusion conditions were applied using a low-viscosity perfusate infused under continuous airway ventilation. Intensely fluorescent polystyrene microspheres, confined to the vascular space, were imaged through confocal optical sectioning of 200 microm-thick lung sections. We applied this technique to rat lungs and the markedly enhanced depth of field in projected images allowed us to follow vascular branching patterns in both normal lungs and lungs from animals with experimentally induced pulmonary arterial hypertension. In addition, this method allowed complementary immunostaining and identification of cellular components surrounding the blood vessels. Fluorescent microangiography is a widely applicable and quantitative tool for the study of vascular changes in animal models of pulmonary disease.

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 alpha, 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 microg 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.

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.

We have investigated the long-term effect of aluminum chloride (AlCl(3)) treatment on the calcification and inflammatory reaction of a porcine elastin-derived biomaterial (PEB) in a novel subdermal adult mouse model. Untreated PEB disks and PEB treated with AlCl(3) were implanted subdermally in BALB/c mice for 30, 60, and 180 days. The calcification of the elastin disks was examined with histological analysis and atomic absorption analysis of calcium content. The inflammatory reaction was evaluated both with histological analysis of explants and by an enzyme-linked immunosorbent assay of the serum in each mouse to determine the production of antielastin antibodies. Robust calcification was evident in all untreated PEBs with calcium levels of 107.1 +/- 11.8, 151.4 +/- 14.4, and 227.2 +/- 23.8 microg/mg for 30, 60, and 180 days, respectively. AlCl(3) treatment only temporarily prevented the calcification of the elastin disks for 30 days. By 60 and 180 days, the AlCl(3)-treated materials had significant calcification with 88.7 +/- 17.4 and 105.3 +/- 27.0 microg/mg calcium, respectively. The inflammatory reaction was moderate for both types of implants. The AlCl(3)-treated implants displayed significantly more macrophage and lymphocyte infiltration at 180 days after implantation, and a trend to higher humoral responses at 30 and 60 days when compared with untreated PEBs. We conclude that PEBs extensively calcify in the adult mice model. AlCl(3) treatment of elastin enhances the long-term immunological response to xenogenic elastin implants and merely delays the onset of calcification., (Copyright 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 55-64, 2004)

Constrictive arterial remodeling accounts for a significant proportion of lumen loss in atherosclerotic progression and postangioplasty stenosis. Recent research suggests that constrictive remodeling is mediated by turnover of the extracellular matrix. We hypothesized that remodeling could be attenuated by treatment with the safe, effective matrix metalloproteinase (MMP) inhibitor doxycycline. Female rabbit abdominal aortas were denuded using a 4-Fr balloon embolectomy catheter, and reinjured 3 weeks later. Treatment with 30 mg/kg/day doxycycline was begun the day before the second injury. At 6 weeks after injury, lumen area measured 13.1 +/- 1.2 mm(2) in controls compared to 17.5 +/- 1.6 mm(2) in doxycycline-treated rabbits (p = 0.05). At 4 days after injury, MMP-2 activity was increased compared to uninjured controls, but doxycycline treatment reduced MMP-2 activity. Doxycycline treatment also inhibited fibrillar collagen deposition in the intima by 87% as detected by polarized light microscopy. Doxycycline was an effective inhibitor of constrictive arterial remodeling in the rabbit aorta. Treatment reduced MMP activity and attenuated the deposition of extracellular matrix particularly in the intima., (Copyright 2004 S. Karger AG, Basel)

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.

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., (Copyright 2001 John Wiley & Sons, Inc. J Biomed Mater Res 55: 576-586, 2001)

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.

We have examined the role of vessel pulsation and wall tension on remodeling and intimal proliferation in the rabbit infrarenal abdominal aorta. A rigid perivascular polyethylene cuff was used to reduce vessel systolic diameter by 25%, producing a region of reduced circumferential strain. At 6 weeks postoperatively, reduced circumferential strain caused medial atrophy, with 45% reduction of medial area and 30% loss of medial smooth muscle cells. Apoptotic cell death was indicated by DNA fragmentation, propidium iodide staining, and cell morphology. Cuffing the aorta after balloon denudation produced medial atrophy but did not inhibit neointimal growth. At 1 week postoperatively, intimal thickness was slightly decreased in regions with reduced strain; however, intimal thickening in regions of reduced strain was not different from control segments at 3 weeks postoperatively (intimal area was 0.37 +/- 0.05 mm2 with reduced strain and 0.50 +/- 0.08 for controls, mean +/- SEM). We conclude that circumferential strain is a major factor controlling medial structure and cell number, whereas growth of the neointima after injury is not significantly affected by either reduced strain or extensive medial cell death. Vessel cuffing represents a new model of blood vessel remodeling in vivo that involves extensive smooth muscle cell apoptosis.

We hypothesized that activation of the coagulation cascade is involved in arterial remodeling in response to sequential injury. An active site-inhibited recombinant human factor VIIa (FVIIai) was used to inhibit tissue factor, the primary cofactor in the extrinsic pathway of coagulation, in a sequential balloon injury model of the rabbit abdominal aorta. Single balloon injury produced limited intimal thickening at 3 weeks (intimal area, 0.40+/-0.05 mm2) and no loss in luminal area (12.2+/-0.9 mm2 before injury and 12.1+/-0.9 mm2 at 6 weeks after injury). Sequential balloon injury, 3 weeks after the first balloon denudation, produced a progressive loss of lumen, with 22% and 47% loss of luminal area, respectively, at 3 and 6 weeks. Luminal loss could not be accounted for by intimal growth (at 3 weeks after sequential injury, the intimal area was 0.47+/-0.08 mm2, <4% of the initial luminal area). Sequential injury acutely produced extensive mural and intramural fibrin deposition. Treatment with FVIIai inhibited both the fibrin deposition and the chronic loss of lumen. At 3 weeks after sequential injury, luminal cross-sectional areas were 9.8+/-0.6 mm2 for control rabbits and 14.3+/-1.4 mm2 for FVIIai-treated rabbits. Neither neointimal area nor cell proliferation was reduced by FVIIai treatment. The intimal cell proliferation index 3 days after injury was 7.6+/-1.1% in control rabbits versus 5.8+/-1.1% in treated rabbits (P>0.05). These results indicate that tissue factor is an important mediator of coagulation in repeat injury and implicate the extrinsic coagulation cascade in a blood vessel remodeling response that is independent of neointimal growth but leads to extensive loss of lumen.

Heart valve substitutes of biological origin often fail by degenerative mechanisms. Many authors have hypothesized that mechanical fatigue and structural degradation are instrumental to in vivo failure. Since the properties of the structural matrix at implantation may predetermine failure, we have examined the ultrastructure, fracture, mechanics, and uniaxial high-strain-rate viscoelastic properties of: (1) fresh, (2) cryopreserved, and (3) cellular extracted porcine aortic valve leaflets. The cellular extraction process is being developed in order to reduce immunological attack and calcification. Cryopreservation causes cellular disruption and necrotic changes throughout the tissue, whereas extraction removes all cells and lipid membranes. Both processes leave an intact collagen and elastin structural matrix and preserve the high-strain-rate viscoelastic characteristics of the fresh leaflets. Extraction does cause a 20% reduction in the fracture tension and increases tissue extensibility, with the percent strain at fracture rising to 45.3 +/- 4 (mean +/- SEM) from 31.5 +/- 3 for fresh leaflets. However, extraction does preserve matrix structure and mechanics over the physiological loading range. Glutaraldehyde fixation produces increased extensibility, increased elastic behavior, and, when applied to extracted leaflets, it causes a marked drop in fracture tension, to 50% of that for fresh leaflets. The combination of extraction and fixation may lead to early degenerative failure. The cellular extraction technique alone may be a useful alternative to glutaraldehyde fixation in preparing bioprosthetic heart valves.

Background: The relation among the coagulation cascade, its individual proteins, and the response to vascular injury is largely undefined. We have evaluated the effect of four probes that block specific levels of coagulation cascade on neointimal hyperplasia in the atherosclerotic rabbit arterial injury model., Methods and Results: Focal femoral atherosclerosis was induced by air-desiccation injury and hypercholesterolemic diet in 48 New Zealand White rabbits, followed by balloon angioplasty. Active-site inactivated factor VIIa (DEGR-VIIa), which blocks the binding of factor VIIa to tissue factor, was administered (n = 12 arteries) by intravenous bolus (1 mg/kg) at the time of balloon angioplasty and followed by infusion of 50 micrograms.kg-1.h-1 for 3 days; for the control (n = 13 arteries), 150 U heparin was injected as bolus and followed by infusion of saline at 50 microL.kg-1.min-1. Recombinant tissue factor pathway inhibitor (TFPI), which binds factor Xa and inhibits the tissue factor-factor VIIa complex and factor Xa, was given as a 1 mg/kg bolus followed by 15 micrograms.kg-1.min-1 infusion for 3 days (n = 17 arteries). Recombinant tick anticoagulant peptide (TAP; n = 15 arteries) and hirudin (n = 14 arteries), which block factor Xa and thrombin, respectively, were administered as a 1 mg/kg bolus followed by 5 micrograms.kg-1.min-1 infusion for 3 days. These three groups had their own controls (n = 14 arteries). There were no differences among treatment groups in preangioplasty and postangioplasty minimal luminal diameter (MLD) by angiography. The mean MLD 21 days after balloon angioplasty was significantly different between control and DEGR-VIIa-treated groups (0.74 +/- 0.25 and 1.24 +/- 0.27 mm, respectively; P = .0001) and between the TFPI-treated group and others (0.88 +/- 0.21 mm for control, 0.97 +/- 0.22 mm for hirudin-treated, 0.98 +/- 0.14 mm for TAP-treated, and 1.32 +/- 0.21 mm for TFPI-treated arteries; P = .0001 by ANOVA). By quantitative histological analysis, the ratio of neointimal cross-sectional area compared with the area of internal elastic lamina in the DEGR-VIIa-treated group was significantly less than control (0.48 +/- 0.12 versus 0.67 +/- 0.12, P = .0001), and the ratio of neointimal cross-sectional area to the area demarcated by the internal elastic lamina of the TFPI-treated group was significantly reduced compared with the other groups (0.46 +/- 0.20 for TFPI-treated, 0.67 +/- 0.15 for hirudin-treated, 0.61 +/- 0.15 for TAP-treated, and 0.64 +/- 0.13 for control groups; P = .003)., Conclusions: Treatment with DEGR-VIIa or TFPI for 3 days in this rabbit atherosclerotic injury model reduced angiographic restenosis and decreased neointimal hyperplasia compared with controls. These findings highlight the importance of early initiators of the extrinsic coagulation pathway, especially factor VII and tissue factor, in the response to arterial injury.

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)

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.

There is evidence to suggest that the cellular components of homografts and bioprosthetic xenografts may contribute to calcification or immunogenic reactions. A four-step detergent and enzymatic extraction process has been developed to remove cellular components from bovine pericardial tissue. The process results in an acellular matrix material consisting primarily of elastin, insoluble collagen, and tightly bound glycosaminoglycans. Light and electron microscopy confirmed that nearly all cellular constituents are removed without ultrastructural evidence of damage to fibrous components. Collagen denaturation temperatures remained unaltered. Biochemical analysis confirmed the retention of collagen and elastin and some differential extraction of glycosaminoglycans. Low strain rate fracture testing and high strain rate viscoelastic characterization showed that, with the exception of slightly increased stress relaxation, the mechanical properties of the fresh tissue were preserved in the pericardial acellular matrix. Crosslinking of the material in glutaraldehyde or poly(glycidyl ether) produced mechanical changes consistent with the same treatments of fresh tissue. The pericardial acellular matrix is a promising approach to the production of biomaterials for heart valve or cardiovascular patching applications.

We have developed an acellular matrix vascular prosthesis (AMVP) made by detergent and enzymatic extraction of natural arteries, yielding a tissue framework of collagen and elastin from the original vessel, with preservation of the natural basement membrane at the blood flow surface. These biografts have excellent handling characteristics and suturability, as well as low thromboreactivity. Whole vessel static testing of circumferential compliance (8.9 +/- 1 [SEM] X 10(-2)% mmHg at 100 mmHg) revealed behavior virtually identical to the paired natural vessel from which each AMVP was derived in nine canine carotid arteries. We implanted 16 canine-origin AMVPs into nine dogs (12 femoral and three carotid arteries, and one infrarenal aorta) with no antithrombotic drugs. Angiographic patency was maintained in 15 of 16 (one occlusion within 3 days) for follow-up from 3 days to 6 years, with no aneurysm formation in three AMVP at over 4 1/2 years. Explant analysis revealed preservation of AMVP elastica and collagen with no inflammation or dystropic calcification of the AMVP, and almost total thrombus free flow surfaces. These results suggest that allograft AMVPs could achieve long-term patency equivalent to saphenous veins.

We have examined the tensile viscoelastic properties of circumferential and radial strips of porcine aortic valve leaflets following fixation in glutaraldehyde and formaldehyde, with or without pressure. After aldehyde treatment, the radial strips remained weaker and less stiff than circumferential strips and responded slightly differently to the treatment. After fixation, with or without pressure, the radial strips showed large changes in stress-strain and hysteresis responses due to initial loading, and there was a twofold reduction in tensile strength and final stiffness. For strips in both directions, fixation without pressure produced doubled extensibility and a ramping stress-strain curve. Permanent (plastic) deformation of 5-20% occurred as a result of cyclic loading, stress relaxation, and creep experiments. Pressure fixation, however, produced little change in stress-strain results other than a simple shift to lower strain and produced no plasticity. Both methods of fixation reduced stress relaxation and creep. Mechanical test results are consistent with a loss of ground substance matrix during fixation. Reductions in tensile strength after fixation may be due to "riveting" of collagen geometry, producing local stress concentrations.

The tensile viscoelastic properties of radial and circumferential strips of fresh porcine aortic valve leaflets have been examined and compared with the fiber architecture of the leaflets as seen in the scanning electron microscope. The leaflet material showed nonlinear viscoelasticity, largely independent of strain rate, and dependent on the degree of preconditioning by cyclic loading. Preconditioning to a stable stress-strain response could only be accomplished on 1-cm-long strips above a minimum width of 4 mm (circumferential) and 6 mm (radial). Preconditioning yielded a more elastic and extensible material with reduced hysteresis. The leaflets were markedly anisotropic. Circumferential strips were up to 8 times stronger and stiffer than radial strips, and displayed greater stress relaxation and less creep. The circumferential mechanical properties were due to well-aligned circumferential collagen bundles reinforcing the composite structure, while radial properties were due to a more random collagenous support throughout the leaflet. Despite the presence of a septal shelf on the right coronary leaflet, no mechanical differences could be discerned between leaflets.