Epicardial transplantation of autologous atrial appendage micrografts: evaluation of safety and feasibility in pigs after coronary artery occlusion.

Objectives. Several approaches devised for clinical utilization of cell-based therapies for heart failure often suffer from complex and lengthy preparation stages. Epicardial delivery of autologous atrial appendage micrografts (AAMs) with a clinically used extracellular matrix (ECM) patch provides a straightforward therapy alternative. We evaluated the operative feasibility and the effect of micrografts on the patch-induced epicardial foreign body inflammatory response in a porcine model of myocardial infarction. Design. Right atrial appendages were harvested and mechanically processed into AAMs. The left anterior descending coronary artery was ligated to generate acute infarction. Patches of ECM matrix with or without AAMs were transplanted epicardially onto the infarcted area. Four pigs received the ECM and four received the AAMs patch. Cardiac function was studied by echocardiography both preoperatively and at 3-week follow-up. The primary outcome measures were safety and feasibility of the therapy administration, and the secondary outcome was the inflammatory response to ECM. Results. Neither AAMs nor ECM patch-related complications were detected during the follow-up time. AAMs patch preparation was feasible according to time and safety. Inflammation was greatly reduced in AAMs when compared with ECM patches as measured by the amount of infiltrated inflammatory cells and area of inflammation. Immunohistochemistry demonstrated an increased CD3+ cell density in the AAMs patch infiltrate. Conclusions. Epicardial AAMs transplantation demonstrated safety and clinical feasibility. The use of micrografts significantly inhibited ECM-induced foreign body inflammatory reactivity. Transplantation of AAMs shows good clinical applicability as adjuvant therapy to cardiac surgery and can suppress acute inflammatory reactivity.

[Beating heart cells--targeted diagnostics, individual optimization of therapy and tailored cell therapies]. / Kantasoluista sykkiviä sydänsoluja.

Myocardial infarction causes scarring and loss of functional capacity of the heart, because the heart is itself unable to repair the damaged area. While the development of new forms of treatment for the repair of myocardial destruction has actually been investigated by introducing into the heart various stem cells present in an adult human, the efficacy of the treatments conducted in the studies has so far unfortunately been low. Embryonic stem cells and iPS cells are a highly significant research subject. Cardiomyocytes differentiated from stem cells are being studied also in drug testing, and they are expected to revolutionize drug development and safety tests of novel drugs as well as enable personalized medication in the future.

Follow-up of intramyocardial bone marrow mononuclear cell transplantation beyond 10 years.

Bone marrow mononuclear cells (BMMCs) have been evaluated for their ability to improve cardiac repair and benefit patients with severe ischemic heart disease and heart failure. In our single-center trial in 2006-2011 we demonstrated the safety and efficacy of BMMCs injected intramyocardially in conjunction with coronary artery bypass surgery. The effect persisted in the follow-up study 5 years later. In this study, we investigated the efficacy of BMMC therapy beyond 10 years. A total of 18 patients (46%) died during over 10-years follow-up and 21 were contacted for participation. Late gadolinium enhancement cardiac magnetic resonance imaging (CMRI) and clinical evaluation were performed on 14 patients, seven from each group. CMRIs from the study baseline, 1-year and 5-years follow-ups were re-analyzed to enable comparison. The CMRI demonstrated a 2.1-fold larger reduction in the mass of late gadolinium enhancement values between the preoperative and the over 10-years follow-up, suggesting less scar or fibrosis after BMMC treatment (- 15.1%; 95% CI - 23 to - 6.7% vs. - 7.3%; 95% CI - 16 to 4.5%, p = 0.039), compared to placebo. No differences in mortality or morbidity were observed. Intramyocardially injected BMMCs may exert long-term benefits in patients with ischemic heart failure. This deserves further evaluation in patients who have received BMMCs in international clinical studies over two decades.

Human skin transcriptome during superficial cutaneous wound healing.

Healing of the epidermis is a crucial process for maintaining the skin's defense integrity and its resistance to environmental threats. Compromised wound healing renders the individual readily vulnerable to infections and loss of body homeostasis. To clarify the human response of reepithelialization, we biopsied split-thickness skin graft donor site wounds immediately before and after harvesting, as well as during the healing process 3 and 7 days thereafter. In all, 25 biopsies from eight patients qualified for the study. All samples were analyzed by genome-wide microarrays. Here, we identified the genes associated with normal skin reepithelialization over time and organized them by similarities according to their induction or suppression patterns during wound healing. Our results provide the first elaborate insight into the transcriptome during normal human epidermal wound healing. The data not only reveal novel genes associated with epidermal wound healing but also provide a fundamental basis for the translational interpretation of data acquired from experimental models.

Heat shock attenuates VEGF expression in three-dimensional myoblast sheets deteriorating therapeutic efficacy in heart failure.

BACKGROUND: Myoblast sheet transplantation is a promising novel treatment for ischemic heart failure. The aim of this study was to test the hypothesis that heat shock (HS) pre-treatment affects the angiogenic properties of myoblast sheets in vivo and in vitro. MATERIAL/METHODS: We studied HS preconditioning of L6 myoblast sheets in relation to their apoptosis, proliferation, and vascular endothelial growth factor (VEGF)-associated responses under normoxia and under hypoxia in vitro. In vivo evaluation of their therapeutic effect was performed with 60 male Wistar rats divided into 3 groups (20 each): sole left anterior descending (LAD) ligation (control); LAD ligation and non-conditioned sheet transplantation (L6 No-Shock); and LAD ligation and L6-heat shock conditioned sheet transplantation (L6 Heat-Shock). Left ventricular function was evaluated by echocardiography after 3, 10, and 28 days. RESULTS: Expression of HSP70/72 was strongly induced 24 hours after HS, and thereafter it decreased notably during 72 hours in hypoxia. Under normal growth conditions, HSP70/72 expression remained stable. HS delayed apoptosis-associated caspase-3 expression during 24-hour hypoxia compared to non-treated controls. However, VEGF expression reduced significantly in the heat shock pretreated sheets. Ejection fraction of the L6-myoblast HS pre-treatment group (L6 Heat-Shock) decreased gradually during follow-up, in the same pattern as the controls. However, these functional parameters improved in the L6-myoblast normal sheet group (L6 No-Shock) at the tenth day and remained significantly better. CONCLUSIONS: HS protects myoblast sheets from hypoxia-associated apoptosis in vitro, but reduces VEGF expression of the sheet, leading to lower therapeutic effect in heart failure.

Ischemic Heart Disease Selectively Modifies the Right Atrial Appendage Transcriptome.

Background: Although many pathological changes have been associated with ischemic heart disease (IHD), molecular-level alterations specific to the ischemic myocardium and their potential to reflect disease severity or therapeutic outcome remain unclear. Currently, diagnosis occurs relatively late and evaluating disease severity is largely based on clinical symptoms, various imaging modalities, or the determination of risk factors. This study aims to identify IHD-associated signature RNAs from the atrial myocardium and evaluate their ability to reflect disease severity or cardiac surgery outcomes. Methods and Results: We collected right atrial appendage (RAA) biopsies from 40 patients with invasive coronary angiography (ICA)-positive IHD undergoing coronary artery bypass surgery and from 8 patients ICA-negative for IHD (non-IHD) undergoing valvular surgery. Following RNA sequencing, RAA transcriptomes were analyzed against 429 donors from the GTEx project without cardiac disease. The IHD transcriptome was characterized by repressed RNA expression in pathways for cell-cell contacts and mitochondrial dysfunction. Increased expressions of the CSRNP3, FUT10, SHD, NAV2-AS4, and hsa-mir-181 genes resulted in significance with the complexity of coronary artery obstructions or correlated with a functional cardiac benefit from bypass surgery. Conclusions: Our results provide an atrial myocardium-focused insight into IHD signature RNAs. The specific gene expression changes characterized here, pave the way for future disease mechanism-based identification of biomarkers for early detection and treatment of IHD.

Epicardial Transplantation of Autologous Cardiac Micrografts During Coronary Artery Bypass Surgery.

Background: Cardio-regenerative cell therapies offer additional biologic support to coronary artery bypass surgery (CABG) and are aimed at functionally repairing the myocardium that suffers from or is damaged by ischemia. This non-randomized open-label study assessed the safety and feasibility of epicardial transplantation of atrial appendage micrografts (AAMs) in patients undergoing CABG surgery. Methods: Twelve consecutive patients destined for CABG surgery were included in the study. Six patients received AAMs during their operation and six patients were CABG-operated without AAMs transplantation. Data from 30 elective CABG patients was collected for a center- and time-matched control group. The AAMs were processed during the operation from a biopsy collected from the right atrial appendage. They were delivered epicardially onto the infarct scar site identified in preoperative late gadolinium enhancement cardiac magnetic resonance imaging (CMRI). The primary outcome measures at the 6-month follow-up were (i) patient safety in terms of hemodynamic and cardiac function over time and (ii) feasibility of therapy administration in a clinical setting. Secondary outcome measures were left ventricular wall thickness, change in myocardial scar tissue volume, changes in left ventricular ejection fraction, plasma concentrations of N-terminal pro-B-type natriuretic peptide levels, NYHA class, number of days in hospital and changes in the quality of life. Results: Epicardial transplantation of AAMs was safe and feasible to be performed during CABG surgery. CMRI demonstrated an increase in viable cardiac tissue at the infarct site in patients receiving AAMs treatment. Conclusions and Relevance: Transplantation of AAMs shows good clinical applicability as performed during cardiac surgery, shows initial therapeutic effect on the myocardium and has the potential to serve as a delivery platform for cardiac gene therapies. Trial Registration:ClinicalTrials.gov, identifier: NCT02672163.

Heat shock enhances troponin expression and decreases differentiation-associated caspase-3 dependence in myoblasts under hypoxia.

BACKGROUND: Myoblast transplantation can functionally restore muscle tissues damaged by ischemic or other insults. Despite promising results in clinical trials, however, myoblast transplantation still presents several challenges, with effective differentiation under harsh conditions of the host tissue being one of the most demanding. In keeping with a straightforward clinical application, heat shock (HS) pretreatment as a nonviral method can be utilized with promising results in cell therapy. The aim of this study was to demonstrate whether HS-pretreated cells would receive a differentiation benefit under hypoxic conditions. MATERIALS AND METHODS: We studied HS preconditioning of C2C12 myoblasts in relation to their differentiation- and apoptosis-associated responses under normoxia or 1% hypoxia. RESULTS: HS induced long-lasting expression of Hsp70/72 and Hsp90. Although myoblast differentiation proceeded in HS-pretreated and control cells under both normoxia and hypoxia, expression of differentiation-associated troponin was enhanced in HS-preconditioned cells under hypoxia. This effect persisted when differentiation was inhibited by Z-DEVD-FMK, a caspase-3 inhibitor. CONCLUSIONS: HS preconditioning enhances expression of myoblast differentiation-associated troponin and may reduce dependence of differentiation on caspase-3.

Epicardial transplantation of atrial appendage micrograft patch salvages myocardium after infarction.

BACKGROUND: Ischemic heart disease remains the leading cause of mortality and morbidity worldwide despite improved possibilities in medical care. Alongside interventional therapies, such as coronary artery bypass grafting, adjuvant tissue-engineered and cell-based treatments can provide regenerative improvement. Unfortunately, most of these advanced approaches require multiple lengthy and costly preparation stages without delivering significant clinical benefits. METHODS: We evaluated the effect of epicardially delivered minute pieces of atrial appendage tissue material, defined as atrial appendage micrografts (AAMs), in a mouse myocardial infarction model. An extracellular matrix patch was used to cover and fix the AAMs onto the surface of the infarcted heart. RESULTS: The matrix-covered AAMs salvaged the heart from the infarction-induced loss of functional myocardium and attenuated scarring. Site-selective proteomics of injured ischemic and uninjured distal myocardium from AAMs-treated and -untreated tissue sections revealed increased expression of several cardiac regeneration-associated proteins (i.e., periostin, transglutaminases, and glutathione peroxidases) and activation of pathways responsible for angiogenesis and cardiogenesis in relation to AAMs therapy. CONCLUSIONS: Epicardial delivery of AAMs encased in an extracellular matrix patch scaffold salvages functional cardiac tissue from ischemic injury and restricts fibrosis after myocardial infarction. Our results support the use of AAMs as tissue-based therapy adjuvants for salvaging the ischemic myocardium.

Bone marrow mesenchymal stem cells undergo nemosis and induce keratinocyte wound healing utilizing the HGF/c-Met/PI3K pathway.

We previously showed cell-cell contacts of human dermal fibroblasts to induce expression of the hepatocyte growth factor/scatter factor (HGF) in a process designated as nemosis. Now we report on nemosis initiation in bone marrow mesenchymal stem cells (BMSCs). Because BMSCs are being used increasingly in cell transplantation therapy we aimed to demonstrate a functional effect and benefit of BMSC nemosis for wound healing. Nemotic and monolayer cells were used to stimulate HaCaT keratinocyte migration in a scratch-wound healing assay. Both indicators of nemosis, HGF production and cyclooxygenase-2 expression, were induced in BMSC spheroids. When compared with a similar amount of cells as monolayer, nemotic cells induced keratinocyte in vitro scratch-wound healing in a concentration-dependent manner. The HGF receptor, c-Met, was rapidly phosphorylated in the nemosis-stimulated keratinocytes. Nemosis-induced in vitro scratch-wound healing was inhibited by an HGF-neutralizing antibody as well as the small molecule c-Met inhibitor, SU11274. HGF-induced in vitro scratch-wound healing was inhibited by PI3K inhibitors, wortmannin and LY294002, while LY303511, an inactive structural analogue of LY294002, had no effect. Inhibitors of the mitogen-activated protein kinases MEK/ERK1/2 (PD98059 and U0126), and p38 (SB203580) attenuated HGF-induced keratinocyte in vitro scratch-wound healing. We conclude that nemosis of BMSCs can induce keratinocyte in vitro scratch-wound healing, and that in this effect signaling via HGF/c-Met is involved.

Spontaneous recovery of myocardial function after ligation of Ameroid-stenosed coronary artery.

OBJECTIVES: We aimed to assess the spontaneous healing of myocardial function after occlusion of a chronically stenosed coronary vessel in a porcine model. DESIGN: Ischemia and infarction was produced by Ameroid constrictor placement and a subsequent ligation of the left circumflex artery. Cardiac MRI and 18FDG-PET were performed one and five weeks later. Ki67 staining was used to identify proliferating cells. RESULTS: Restoration of perfusion defect was detected by MRI (p=0.0065), reduced systolic function of the lateral segment spontaneously recovered (p=0.03). There was also a suggestive raise in impaired ejection fraction (p=0.06). Left ventricular early diastolic filling and peak filling rate were substantially improved (p=0.039 and p=0.0078). Scar size reduced (p=0.03). On the 18FDG-PET, deranged metabolism was alleviated (p=0.03). Cardiomyocytes with positive Ki-67 staining were located principally in the non-infarcted myocardium as compared to the infarction or border areas (p=0.037). CONCLUSIONS: We demonstrated spontaneous functional healing of ischemic and infarcted left ventricle, suggesting border zone perfusion recovery. Scar reduction was detected. Different pattern of myocyte proliferation between infarction and non-ischemic myocardium was seen.

Improved diastolic function after myoblast transplantation in a model of ischemia-infarction.

OBJECTIVES: The aim of this study was to evaluate the effect of myoblast transplantation on left ventricular function, perfusion, and scar formation after compromised coronary flow. DESIGN: A coronary vessel with Ameroid-induced stenosis was ligated and skeletal muscle was biopsied for isolation and cultivation of myoblasts. Two weeks after ligation, animals were randomly selected to receive intramyocardial injections of 2 x 10(6) myoblasts or vehicle. Fifteen animals survived the whole study period (n=9 and n=6, respectively). All animals underwent cardiac magnetic resonance imaging (MRI) and angiography pretreatment and four weeks posttreatment. RESULTS: Peak filling rate of the left ventricle improved in the myoblast group (p=0.0048), but not in the control group. Peak ejection rate and duration of diastole improved only in the myoblast group (p=0.049 and p=0.0039, respectively). Ejection fraction or local thickening did not change. Fibrosis and perfusion were similar in both groups, but more microvessels were present histologically in the myoblast group. CONCLUSIONS: In this preclinical study, autologous myoblast transplantation improved ischemic heart function via enhanced diastolic filling of the left ventricle.

Fibroblast nemosis arrests growth and induces differentiation of human leukemia cells.

Interactive signaling between cancer cells and stroma plays an important role in determining tumor development. We recently found tumor cell-derived factors to induce fibroblast clustering, and that the high amounts of hepatocyte growth factor/scatter factor (HGF/SF) produced by these cell-cell contact-activated fibroblasts enhanced the invasiveness of c-Met-expressing cancer cells. We now observed that leukemia cells lacking c-Met respond to this novel type of fibroblast activation, nemosis, with growth arrest and differentiation to a dendritic cell-like phenotype. This effect was counteracted by introduction of c-Met expression into these cells. Moreover, those leukemia cell lines harboring properly processed c-Met showed no effect in response to nemosis. We propose this effect to be mediated by nemosis-derived cytokine signals, and present the potential candidates induced in the nemotic fibroblasts: interleukins-1, -6, -8, -11, leukemia inhibitory factor and granulocyte-macrophage-colony-stimulating factor. Our results emphasize the role of activated stromal fibroblasts in controlling progression of certain hematologic malignancies in a c-Met expression-dependent manner.

Persistent susceptibility of cathepsin B to irreversible inhibition by nitroxyl (HNO) in the presence of endogenous nitric oxide.

Nitrosation of enzyme regulatory cysteines is one of the key posttranslational modification mechanisms of enzyme function. Frequently such modifications are readily reversible; however, cysteine proteases, such as cathepsin B, have been shown to be covalently and permanently inactivated by nitroxyl (HNO), the one-electron reduction product of NO. Owing to the high reactivity of HNO with NO, endogenous NO production could provide direct protection for the less reactive protein cysteines by scavenging HNO. Additionally, endogenous cellular production of NO could rescue enzyme function by protective nitrosation of cysteines prior to exposure to HNO. Thus, we studied the effect of endogenous NO production, induced by LPS or IFN-gamma, on inhibition of cysteine protease cathepsin B in RAW macrophages. Both LPS and IFN-gamma induce iNOS with generation of nitrate up to 9 muM in the media after a 24-h stimulation, while native RAW 264.7 macrophages neither express iNOS nor generate nitrate. After the 24-h stimulation, the HNO-releasing Angeli's salt (0-316 microM) caused dose-dependent and DTT-irreversible loss of cathepsin B activity, and induction of iNOS activity did not protect the enzyme. The lack of protection was also verified in an in vitro setup, where papain, a close structural analogue of cathepsin B, was inhibited by Angeli's salt (2.7 microM) in the presence of the NO donor DEA/NO (0-316 microM). This clearly showed that a high molar excess of DEA/NO (EC(50) 406 microM) is needed to protect papain from the DTT-irreversible covalent modification by HNO. Our results provide first evidence on a cellular level for the remarkably high sensitivity of active-site cysteines in cysteine proteases for modification by HNO.

Polyglycolic acid glue does not prevent intrapericardial adhesions in a short-term follow-up.

BACKGROUND: Pericardial adhesions are a significant challenge for reoperative cardiac surgery. There are no established means of prevention. Lately, sprayable glues have been suggested to inhibit and reduce adhesion formation in congenital heart surgery, where repeated cardiac operations are often needed. In this study, we tested a novel, synthetic, sprayable, and resorbable polyglycolic acid glue (DuraSeal; Confluent Surgical, Inc., Waltham, MA) that has been approved by the United States Food and Drug Administration as a dural sealant. MATERIAL AND METHODS: A standard sternotomy and longitudinal pericardiotomy was performed in seven pigs, and polyglycolic acid glue (DuraSeal) was administered randomly on either side of the heart. The pericardium was sutured and autologous blood was instilled into pericardium to augment adhesion formation. After 6 wk, the hearts were photographed and examined in terms of adhesion formation (tenacity, extension, and strength), and visibility of the coronary vessels. A semiquantitative scale 0-3 was used. RESULTS: All animals produced significant pericardial adhesions. There were no statistically significant differences in tenacity, extension, or strength of adhesions between glued and non-glued sides of the hearts, nor was there any significant variation in the visibility of the coronary vessels between the sides. CONCLUSIONS: The DuraSeal polyglycolic acid glue did not reduce the development of pericardial adhesions in pigs.

Epicardial delivery of autologous atrial appendage micrografts during coronary artery bypass surgery-safety and feasibility study.

BACKGROUND: The atrial appendages are a tissue reservoir for cardiac stem cells. During on-pump coronary artery bypass graft (CABG) surgery, part of the right atrial appendage can be excised upon insertion of the right atrial cannula of the heart-lung machine. In the operating room, the removed tissue can be easily cut into micrografts for transplantation. This trial aims to assess the safety and feasibility of epicardial transplantation of atrial appendage micrografts in patients undergoing CABG surgery. METHODS/DESIGN: Autologous cardiac micrografts are made from leftover right atrial appendage during CABG of 6 patients. Atrial appendage is mechanically processed to micrografts consisting of atrial appendage-derived cells (AADCs) and their extracellular matrix (ECM). The micrografts are epicardially transplanted in a fibrin gel and covered with a tissue-engineered ECM sheet. Parameters including echocardiography-reflecting cardiac insufficiency-are studied pre- and post-operatively as well as at 3 and 6 months of the follow-up. Cardiac functional magnetic resonance imaging is performed preoperatively and at 6-month follow-up. The primary outcome measures are patient safety in terms of hemodynamic and cardiac function over time and feasibility of therapy administration in a clinical setting. Secondary outcome measures are left ventricular wall thickness, change in the amount of myocardial scar tissue, changes in left ventricular ejection fraction, plasma concentrations of N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels, New York Heart Association class, days in hospital, and changes in the quality of life. Twenty patients undergoing routine CAGB surgery will be recruited to serve as a control group. DISCUSSION: This study aims to address the surgical feasibility and patient safety of epicardially delivered atrial appendage micrografts during CABG surgery. Delivery of autologous micrografts and AADCs has potential applications for cell and cell-based gene therapies. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02672163. Date of registration: 02.02.2016.

Rational Autologous Cell Sources For Therapy of Heart Failure - Vehicles and Targets For Gene and RNA Therapies.

This review focuses on the possibilities for intraoperative processing and isolation of autologous cells, particularly atrial appendage-derived cells (AADCs) and cellular micrografts, and their straightforward use in cell transplantation for heart failure therapy. We review the potential of autologous tissues to serve as sources for cell therapy and consider especially those tissues that are used in surgery but from which the excess is currently discarded as surgical waste. We compare the inculture expanded cells to the freshly isolated ones in terms of evidence-based cost-efficacy and their usability as gene- and RNA therapy vehicles. We also review how financial and authority-based decisions and restrictions sculpt the landscape for patients to participate in academic-based trials. Finally, we provide an insight example into AADCs isolation and processing for epicardial therapy during coronary artery bypass surgery.

The Paracrine Effect of Skeletal Myoblasts Is Cardioprotective Against Oxidative Stress and Involves EGFR-ErbB4 Signaling, Cystathionase, and the Unfolded Protein Response.

Therapeutic effects of skeletal myoblast transplantation into the myocardium are mediated via paracrine factors. We investigated the ability of myoblast-derived soluble mediators to protect cardiomyocytes from oxidative stress. Fetal rat cardiac cells were treated with conditioned medium from cultures of myoblasts or cardiac fibroblasts, and oxidative stress was induced with H2O2. Myoblast-derived factors effectively prevented oxidative stress-induced cardiac cell death and loss of mitochondrial membrane potential. This protective effect was mediated via epidermal growth factor (EGF) receptor and c-Met signaling, and mimicked by neuregulin 1 but not EGF. Microarray analysis of cardiac cells treated with myoblast versus cardiac fibroblast-derived mediators revealed differential regulation of genes associated with antioxidative effects: cystathionine-γ-lyase (cst), xanthine oxidase, and thioredoxin-interacting protein as well as tribbles homolog 3 (trib3). Cardiac cell pretreatment with tunicamycin, an inducer of trib3, also protected them against H2O2-induced cell death. Epicardial transplantation of myoblast sheets in a rat model of acute myocardial infarction was used to evaluate the expression of CST and trib3 as markers of myoblasts' paracrine effect in vivo. Myoblast sheets induced expression of the CST as well as trib3 in infarcted myocardium. CST localized around blood vessels, suggesting smooth muscle cell localization. Our results provide a deeper molecular insight into the therapeutic mechanisms of myoblast-derived paracrine signaling in cardiac cells and suggest that myoblast transplantation therapy may prevent oxidative stress-induced cardiac deterioration and progression of heart failure.

Matrix metalloproteinase induction in post-transplant obliterative bronchiolitis.

BACKGROUND: Epithelial cell injury, inflammation, fibrosis, and airway obliteration are associated in post-transplant obliterative bronchiolitis. Fibrosis is a consequence of fibroblastic activity and of collagen deposition after disturbances in the balance of protein formation and degradation. Proteolytic enzymes such as the matrix metalloproteinases mediate degradation. To assess matrix metalloproteinases during obliterative bronchiolitis development, we studied porcine, heterotopic bronchial allografts. METHODS: A total of 119 allografts or autografts were harvested serially at 3 to 60 days after transplantation and processed for histology and in situ hybridization for matrix metalloproteinases 2 and 9. Immunocytochemistry for vimentin and alpha-smooth-muscle-cell actin was performed with specific antibodies. RESULTS: Implants had initial ischemic injury to airway epithelium and to the bronchial wall. Recovery was rapid in autografts and in immunosuppressed allografts. In matrix metalloproteinase-2 mRNA activity in fibroblasts, correlation with endothelial expression and expression in macrophages occurred during intense fibroproliferation. We observed intense matrix metalloproteinase-9 positivity during onset of inflammation and fibroproliferation in endothelial cells (p < 0.01), fibroblasts (p < 0.05), macrophages (p < 0.05), and lymphocytes (p < 0.05). Matrix metalloproteinase-9 mRNA activity in fibroblasts correlated with that in endothelial and inflammatory cells and also proved predictive of early obliteration. CONCLUSIONS: Matrix metalloproteinase-2, and especially matrix metalloproteinase-9, gene activity was associated with onset of inflammation and fibroblastic proliferation in allografts, predicting early obliteration. Although this may be the case in the model described, its role in human-allograft post-transplant obliterative bronchiolitis requires further supportive data.

Recombinant human collagen III gel for transplantation of autologous skin cells in porcine full-thickness wounds.

Complex skin wounds, such as chronic ulcers and deep burns, require lengthy treatments and cause extensive burdens on healthcare and the economy. Use of biomaterials and cell transplantation may improve traditional treatments and promote the healing of difficult-to-treat wounds. In this study, we investigated the use of recombinant human collagen III (rhCol-III) gel as a delivery vehicle for cultured autologous skin cells (keratinocytes only or keratinocyte-fibroblast mixtures). We examined its effect on the healing of full-thickness wounds in a porcine wound-healing model. Two Landrace pigs were used for the study. Fourteen deep dermal wounds were created on the back of each pig with an 8 mm biopsy punch. Syringes containing acellular rhCol-III gel (n = 8) or rhCol-III gel with autologous keratinocytes (n = 8) or rhCol-III gel with autologous keratinocytes and fibroblasts (n = 8) were applied into wounds. Untreated wounds were used as controls for the treatment groups (n = 4). We used rhCol-III gel to manufacture a cell-delivery syringe containing autologous skin cells. In a full-thickness wound-healing model, we observed that rhCol-III gel enhances early granulation tissue formation. Interestingly, we found cell type-dependent differences in the stability of rhCol-III in vivo. Fibroblast-containing gel was effectively removed from the wound, whereas gels without cells or with keratinocytes only remained intact. Our results demonstrate that the properties of rhCol-III gel for skin cell transplantation can be significantly altered in a cell type-dependent manner.