Kristopher B. Deatrick

Postthrombotic vein wall remodeling: preliminary observations.

BACKGROUND Postthrombotic syndrome is characterized by a fibrotic vein injury following deep vein thrombosis (DVT). We sought to quantify the change in vein wall thickness in patients who fail to resolve DVT by 6 months and whether there were differences in blood or plasma levels of inflammatory proteins associated with venous remodeling. METHODS Patients presenting with confirmed lower extremity DVT were prospectively recruited for this study. Duplex imaging of the lower extremity venous system was performed, and blood was collected at entrance and repeat evaluation with blood draw and ultrasound imaging at 1 and 6 months. DVT resolution and thickness of the vein wall was quantified by ultrasound imaging in each segment affected by thrombus, and a contralateral, unaffected vein wall served as a control. Gene and protein expression of inflammatory markers were examined from leukocytes and serum, respectively. Analysis of variance or Student t-tests were used, and a P < .05 was significant. N = 10 to 12 for all analyses. RESULTS Thirty-two patients (12 patients with DVT resolution at 6 months, 10 patients with persistent thrombus at 6 months, and 10 healthy controls) were compared. Both resolving and nonresolving DVT were associated with a 1.5- to 1.8-fold increased vein wall thickness at 6 months (P = .008) as compared with nonaffected vein wall segments. However, the thickness of the affected segments was 1.4-fold greater in patients who had total resolution of the DVT by 6 months than in patients who had persistent chronic thrombus 6 months after presentation (P = .01). There was a four- to five-fold increased level of matrix metalloproteinase-9 (MMP-9) antigen in thrombosed patients compared with nonthrombosed patient controls (P < .05), while Toll-like receptor-9 (TLR-9) gene expression was three-fold less than controls (P < .05) at enrollment. D-dimer and P-selectin were higher in thrombosed as compared to controls at diagnosis but not at 6 months. Both TLR-4 (marker of inflammation) and P-selectin gene expression were higher in leukocytes from patients with chronic DVT compared with those who resolved at 1 month after diagnosis (P < .05). CONCLUSIONS This preliminary study suggests ongoing vein wall remodeling after DVT, measurable by ultrasound and associated with certain biomarkers. At 6 months, the vein wall is markedly thickened and directly correlates with resolution. This suggests that the vein wall response is initiated early following thrombus formation and persists even in the presence of total resolution.

Urokinase plasminogen activator independent early experimental thrombus resolution: MMP2 as an alternative mechanism

Deep-vein thrombosis (DVT) resolution is thought to be primarily a urokinase plasminogen activator (uPA) -dependent mechanism, although observations suggest other non-fibrinolytic mechanisms may exist. We explored the role of matrix metalloproteinase (MMP) -2 and -9 in early DVT resolution in uPA-deficient mice. Male B6/SVEV (WT) and genetically matched uPA -/- mice underwent inferior vena cava (IVC) ligation to create stasis venous thrombi, with IVC and thrombus harvest. Thrombus size was similar between WT and uPA -/- mice at day 4, suggesting early non uPA-dependent resolution. Intrathrombus neutrophils and monocytes were reduced 3- and 3.5-fold in uPA -/- mice as compared with WT. By ELISA, tumour necrosis factor α and interleukin 1β were not altered, while interferon (IFN)γ was significantly elevated in uPA -/- mice. A compensatory increase in thrombus tPA was not observed, plasmin activity was reduced and PAI-1 was elevated 2.5-fold in uPA -/- mice. Active MMP2, but not MMP9, was elevated 3-fold in uPA -/- mice as compared with WT as well as MMP-14, an MMP2 activator. Collagen type IV and fibrinogen were reduced in uPA -/- mice thrombi as compared with WT. IFNγ induces MMP2, and blockade of IFNγ was associated with larger venous thrombi and reduced active MMP2, as compared with WT. Consistently, MMP2 -/- mice had larger VT as compared with WT controls, despite normal thrombus plasmin levels. Taken together, early experimental venous thrombus resolution is independent of uPA, and, in part, inflammatory cell influx. MMP2-dependent thrombolysis is an important compensatory mechanism of venous thrombus resolution, possibly by collagen type IV metabolism, and may represent an exploitable therapeutic avenue.

The effect of matrix metalloproteinase 2 and matrix metalloproteinase 2/9 deletion in experimental post-thrombotic vein wall remodeling

BACKGROUND Vein wall fibrotic injury following deep venous thrombosis (VT) is associated with elevated matrix metalloproteinases (MMPs). Whether and by what mechanism MMP2 contributes to vein wall remodeling after VT is unknown. METHODS Stasis VT was produced by ligation of the inferior vena cava and tissue was harvested at 2, 8, and 21 days in MMP2 -/- and genetic wild type (WT) mice. Tissue analysis by immunohistochemistry, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and zymography was performed. RESULTS Thrombus resolution was less at 8 days in MMP2 -/- compared with WT, evidenced by a 51% increase in VT size (P < .01), and threefold fewer von Willebrands factor positive channels (P < .05). In MMP2 -/- mice, the main phenotypic fibrotic differences occurred at 8 days post-VT, with significantly less vein wall collagen content (P = .013), fourfold lower procollagen III gene expression (P < .01), but no difference in procollagen I compared with WT. Decreased inflammation in MMP2 -/- vein walls was suggested by ∼ threefold reduced TNFα and IL-1β at 2 days and 8 days post-VT (P < .05). A fourfold increase in vein wall monocytes (P = .03) with threefold decreased apoptosis (P < .05), but no difference in cellular proliferation at 8 days was found in MMP2 -/- compared with WT. As increased compensatory MMP9 activity was observed in the MMP2 -/-mice, MMP2/9 double null mice had thrombus induced with VT harvest at 8 days. Consistently, twofold larger VT, a threefold decrease in vein wall collagen, and a threefold increase in monocytes were found (all P < .05). Similar findings were observed in MMP9 -/- mice administered an exogenous MMP2 inhibitor. CONCLUSIONS In stasis VT, deletion of MMP2 was associated with less midterm vein wall fibrosis and inflammation, despite an increase in monocytes. Consideration that VT resolution was impaired with MMP2 (and MMP2/9) deletion suggests direct inhibition will likely also require anticoagulant therapy.

Matrix metalloproteinase-9 deletion is associated with decreased mid-term vein wall fibrosis in experimental stasis DVT.

INTRODUCTION Post thrombotic syndrome therapy is primarily palliative, and the associated vein wall inflammatory mechanisms are unclear. Vein wall fibrotic injury following deep venous thrombosis (VT) is associated with elevated matrix metalloproteinases (MMPs). Whether and by what mechanism MMP9 directly contributes to vein wall remodeling after VT is unknown. METHODS WT and MMP9 -/- mice underwent stasis VT by ligation of the inferior vena cava (IVC) and tissue was harvested at 2, 8, and 21days. Assessment of thrombus size, and gene, protein and structural vein wall determinations were done. RESULTS VT resolution was increased in MMP9-/- mice as compared with controls at 21d only. The primary phenotypic fibrotic vein wall differences occurred at 8d post VT, with significantly less vein wall collagen content as assessed by Picosirius red staining in MMP9 -/- mice as compared with WT. Increased monocytic vein wall influx with less IL-1b and TGFb was found in MMP9 -/- vein walls as compared with WT. Corresponding levels of PAI-1 were increased in MMP9 -/- compared with WT, and no difference in FSP-1+cells as compared with controls. CONCLUSIONS In stasis VT, MMP9 modulates midterm vein wall collagen content, with an altered local inflammatory and profibrotic environment, likely directed by monocytes. Thus, MMP9 plays a role in both vein wall responses as well as late thrombus resolution.

Evidence-based management of chylothorax in infants

PURPOSE Management guidelines for infants with chylothorax lack substantial evidence. We sought to identify variables that impact outcomes in these patients in order to develop an evidence-based management algorithm. METHODS We retrospectively reviewed the medical records of all infants diagnosed with chylothorax from June 2005 to December 2014 at our institution. Data collected included demographics, chest tube output (CTO), medical and dietary interventions, surgical procedures, and absolute lymphocyte count (ALC). Outcomes analyzed included death, sepsis, necrotizing enterocolitis (NEC), requiring surgery, and success of therapy, defined as CTO decrease by >50% within 7days. RESULTS Of 178 neonates with chylothorax, initial therapy was high medium chain triglyceride (MCT) feedings in 106 patients, nothing by mouth (NPO), total parenteral nutrition (TPN) in 21, and NPO/TPN plus octreotide in 45. Octreotide use in addition to NPO/TPN revealed no significant differences in any outcome including success (47% vs. 43%, p=0.77). Initial CTO and ALC correlated with needing surgery (p=0.002 and p=0.006, respectively), and with death (p=0.028 and p=0.043, respectively). ALC also correlated with sepsis (p<0.001). CONCLUSIONS Octreotide has no advantage over NPO/TPN alone in infants with chylothorax. CTO and ALC predict requiring surgery. We propose a management guideline based on CTO and ALC without a role for octreotide. TYPE OF STUDY Retrospective case-control study. LEVEL OF EVIDENCE 3.

Current and future status of extracorporeal life support for respiratory failure in adults

Purpose of review The purpose is to review the development and current application of extracorporeal life support [ECLS, extracorporeal membrane oxygenation (ECMO)] in acute severe respiratory failure. Recent findings Extracorporeal support (ECMO) is used for acute severe respiratory failure in advanced ICUs. The current survival rate is 60–70%. Three controlled trials all demonstrated 20–30% improvement in survival compared to conventional care. Patients may now be maintained awake and ambulatory with spontaneous breathing. Summary ECMO is the next step in the algorithm for management of severe respiratory failure unresponsive to conventional care.

Stem Cell Therapy for Hypoplastic Left Heart Syndrome: Mechanism, Clinical Application, and Future Directions

Hypoplastic left heart syndrome is a type of congenital heart disease characterized by underdevelopment of the left ventricle, outflow tract, and aorta. The condition is fatal if aggressive palliative operations are not undertaken, but even after the complete 3-staged surgical palliation, there is significant morbidity because of progressive and ultimately intractable right ventricular failure. For this reason, there is interest in developing novel therapies for the management of right ventricular dysfunction in patients with hypoplastic left heart syndrome. Stem cell therapy may represent one such innovative approach. The field has identified numerous stem cell populations from different tissues (cardiac or bone marrow or umbilical cord blood), different age groups (adult versus neonate-derived), and different donors (autologous versus allogeneic), with preclinical and clinical experience demonstrating the potential utility of each cell type. Preclinical trials in small and large animal models have elucidated several mechanisms by which stem cells affect the injured myocardium. Our current understanding of stem cell activity is undergoing a shift from a paradigm based on cellular engraftment and differentiation to one recognizing a primarily paracrine effect. Recent studies have comprehensively evaluated the individual components of the stem cells’ secretomes, shedding new light on the intracellular and extracellular pathways at the center of their therapeutic effects. This research has laid the groundwork for clinical application, and there are now several trials of stem cell therapies in pediatric populations that will provide important insights into the value of this therapeutic strategy in the management of hypoplastic left heart syndrome and other forms of congenital heart disease. This article reviews the many stem cell types applied to congenital heart disease, their preclinical investigation and the mechanisms by which they might affect right ventricular dysfunction in patients with hypoplastic left heart syndrome, and finally, the completed and ongoing clinical trials of stem cell therapy in patients with congenital heart disease.

Study design and rationale for ELPIS: A phase I/IIb randomized pilot study of allogeneic human mesenchymal stem cell injection in patients with hypoplastic left heart syndrome

Despite advances in surgical technique and postoperative care, long-term survival of children born with hypoplastic left heart syndrome (HLHS) remains limited, with cardiac transplantation as the only alternative for patients with failing single ventricle circulations. Maintenance of systemic right ventricular function is crucial for long-term survival, and interventions that improve ventricular function and avoid or defer transplantation in patients with HLHS are urgently needed. We hypothesize that the young myocardium of the HLHS patient is responsive to the biological cues delivered by bone marrow-derived mesenchymal stem cells (MSCs) to improve and preserve right ventricle function. The ELPIS trial (Allogeneic Human MEsenchymal Stem Cell Injection in Patients with Hypoplastic Left Heart Syndrome: An Open Label Pilot Study) is a phase I/IIb trial designed to test whether MSC injection will be both safe and feasible by monitoring the first 10 HLHS patients for new major adverse cardiac events. If our toxicity stopping rule is not activated, we will proceed to the phase IIb component of our study where we will test our efficacy hypothesis that MSC injection improves cardiac function compared with surgery alone. Twenty patients will be enrolled in a randomized phase II trial with a uniform allocation to MSC injection versus standard surgical care (no injection). The 2 trial arms will be compared with respect to improvement of right ventricular function, tricuspid valve annulus size, and regurgitation determined by cardiac magnetic resonance and reduced mortality, morbidity, and need for transplantation. This study will establish the safety and feasibility of allogeneic mesenchymal stem cell injection in HLHS patients and provide important insights in the emerging field of stem cell-based therapy for congenital heart disease patients.

Mechanical Circulatory Support for the Failing Fontan: Conversion to Assisted Single Ventricle Circulation—Preliminary Observations

Background: Mechanical circulatory support (MCS) of a failing Fontan circulation remains challenging. We hypothesized that MCS can be provided by converting the Fontan circulation into a mechanically assisted single ventricle parallel circulation (MASVC). Methods: A porcine model of functionally univentricular circulation was created under cardiopulmonary bypass (CPB) by performing an atrial septectomy, tricuspid valvectomy, and interrupting antegrade pulmonary blood flow. A centrifugal flow pump was placed with inflow from the common atrium. Eight millimeter Dacron grafts anastomosed to the ascending aorta and main pulmonary artery supplied systemic (Qs) and pulmonary (Qp) blood flow. Ultrasonic flow probes were used to measure Qs and Qp after weaning from CPB. The Qp/Qs ratio was regulated using an adjustable clamp. Hemodynamic and laboratory data were recorded. Results: All four animals were successfully weaned from CPB onto the MASVC for a duration of two hours. Mechanically assisted single ventricle parallel circulation achieved satisfactory hemodynamics. As anticipated, the arterial oxygen saturation and partial pressure of oxygen in arterial blood were lower in the MASVC compared to baseline biventricular circulation. At the conclusion of the study, there was a trend towards a decrease in the mixed venous saturation with increasing oxygen extraction compared to the baseline. Serum lactate levels increased after weaning from CPB and did not return to baseline after two hours of support. Conclusion: Mechanically assisted single ventricle parallel circulation can be established in a single ventricle animal model. This strategy could potentially provide MCS of a single ventricle circulation. Studies with longer duration of support are required to assess adequacy of support and long-term sustainability.

Differential effects of normoxic and hyperoxic reperfusion on global myocardial ischemia-reperfusion injury

The objectives were to investigate if after hypoxia or ischemia, normoxic reperfusion is associated with less oxidant stress (OS), inflammation, and myocardial injury than hyperoxic reperfusion. In this study, cardiomyocytes (H9c2 cells) were cultured in hypoxia, followed by reoxygenation in normoxia or hyperoxia. Cardiomyocyte OS, inflammation, and apoptosis were measured. In parallel experiments, rabbits were cannulated for cardiopulmonary bypass (CPB). Following cardioplegic arrest and aortic cross-clamp removal, hearts were reperfused under normoxic or hyperoxic conditions. Left ventricular developed pressure and contractility (LV +dP/dt) were recorded, and blood samples and heart tissues were collected for measurement of OS, inflammation, and cardiac injury. Results showed that H9c2 cells exposed to hyperoxic reoxygenation showed significant increases in OS, inflammation, and apoptosis compared to normoxic reoxygenation. Following CPB and 2-hour hyperoxic reperfusion, LV +dP/dt and left ventricular developed pressure were significantly decreased compared with pre-CPB values (to 36 ± 21%, P = 0.002; and 53 ± 20%, P = 0.02, respectively), associated with significant increases in all plasma and tissue biomarkers for OS, inflammation, and myocardial injury. In contrast, LV +dP/dt was relatively well preserved under normoxic reperfusion conditions (to 70 ± 14% after 2-hour reperfusion), and was associated with an attenuated myocardial OS, inflammatory, apoptotic, and injury response compared to the hyperoxia group (eg, cTn-I: 5.9 ± 1.5 vs 20.2 ± 7.6 ng/mL, respectively, P < 0.0001). Overall, in both in vitro and in vivo experiments, normoxic reperfusion/reoxygenation was associated with less robust OS, inflammation, apoptosis, and myocardial injury compared with hyperoxic reperfusion/reoxygenation. These results suggest that hyperoxia should be avoided to minimize myocardial OS, inflammation, and ventricular dysfunction after CPB.