Ashok Babu

Microvascular destruction identifies murine allografts that cannot be rescued from airway fibrosis

Small airway fibrosis (bronchiolitis obliterans syndrome) is the primary obstacle to long-term survival following lung transplantation. Here, we show the importance of functional microvasculature in the prevention of epithelial loss and fibrosis due to rejection and for the first time, relate allograft microvascular injury and loss of tissue perfusion to immunotherapy-resistant rejection. To explore the role of alloimmune rejection and airway ischemia in the development of fibroproliferation, we used a murine orthotopic tracheal transplant model. We determined that transplants were reperfused by connection of recipient vessels to donor vessels at the surgical anastomosis site. Microcirculation through the newly formed vascular anastomoses appeared partially dependent on VEGFR2 and CXCR2 pathways. In the absence of immunosuppression, the microvasculature in rejecting allografts exhibited vascular complement deposition, diminished endothelial CD31 expression, and absent perfusion prior to the onset of fibroproliferation. Rejecting grafts with extensive endothelial cell injury were refractory to immunotherapy. After early microvascular loss, neovascularization was eventually observed in the membranous trachea, indicating a reestablishment of graft perfusion in established fibrosis. One implication of this study is that bronchial artery revascularization at the time of lung transplantation may decrease the risk of subsequent airway fibrosis.

Lipopolysaccharide Stimulation of Human Aortic Valve Interstitial Cells Activates Inflammation and Osteogenesis

BACKGROUND Calcific aortic stenosis may be an inflammatory disease with active bone formation in the valve leaflets rather than a disease of passive calcium deposition. Epidemiologic data demonstrating correlation of poor dental hygiene to atherosclerotic pathologies suggests that circulating bacterial products could be involved in the pathogenesis of aortic valve stenosis. We hypothesized that lipopolysaccharide (LPS) stimulation of human aortic valve interstitial cells (HAVICs) would induce inflammatory and osteogenic gene expression. METHODS The HAVICs were isolated from normal aortic valves obtained from explanted hearts during transplantation (n = 5) and grown in culture. Cells underwent 4 and 24 hours of LPS stimulation (LPS, 200 ng/mL) or beta-glycerol phosphate treatment (BGP) (osteogenic media as positive control). Media was removed for interleukin (IL)-6 and IL-8 immunoassay. Ribonucleic acid was extracted for microarray analysis. Statistics were by analysis of variance with post-hoc analysis (p < 0.05). RESULTS The LPS stimulation induced the gene expression of proinflammatory cytokines, chemokines, and adhesion molecules. Protein level confirmation by immunoassay demonstrated 3.4-fold (+/- 0.35, p < 0.01) and 9.5-fold (+/- 1.5 p < 0.01) increase over control of IL-6 and IL-8, respectively. The LPS and BGP both induced critical mediators of osteogenesis including bone morphogenetic protein 2 and platelet-derived growth factor alpha. CONCLUSIONS The LPS stimulation of HAVICs not only induces inflammatory mediators but also induces gene expression of osteogenic factors, similar to that induced by osteogenic media. Bacterial products stimulation, likely by toll-like receptor 4 and the innate immune system, may contribute to the pathogenesis of aortic valve stenosis.

Changes in Aortic Wall Structure, Composition, and Stiffness With Continuous-Flow Left Ventricular Assist Devices: A Pilot Study

Background—The effects of nonpulsatile flow on the aorta are unknown. Our aim was to examine the structure of the aorta from patients with continuous-flow left ventricular assist devices (LVADs) and directly measure aortic wall composition and stiffness. Methods and Results—Age-matched aortic wall samples were collected from consecutive patients with heart failure (HF) at the time of transplantation and compared with nonfailing donor hearts. An unbiased stereological approach was used to quantify aortic morphometry and composition, and biomechanical testing was performed to determine the stress–strain relationship of the vessel. Data were obtained from 4 patients without a left ventricular assist device (HF group: mean age, 58.3±8.0 years), 7 patients with a continuous-flow LVAD (HF+LVAD group: mean, 57.7±5.6 years), and 3 nonfailing donors (mean, 53.3±12.9 years). Compared with HF, the aortic walls from HF+LVAD had an increase in wall thickness, collagen, and smooth muscle content accompanied by a reduction in elastin and mucinous ground-substance content. Stress–strain curves from the aortas revealed increased vessel stiffness in HF+LVAD compared with HF and nonfailing. The physiological modulus of the aorta progressively stiffened from 74.3±5.5 kPa in the nonfailing to 134.4±35.0 kPa in the HF to 201.7±36.4kPa in the HF+LVAD groups (P<0.001). Conclusions—Among continuous-flow LVAD patients without aortic valve opening, there are changes in the structure and composition of the aorta as well as an increase in aortic wall stiffness compared with age-matched HF patients and nonfailing donors. Further studies examining the role of nonpulsatile blood flow on aortic function and the potential resultant systemic sequelae are needed.

Secretory phospholipase A2 is required to produce histologic changes associated with gastroduodenal reflux in a murine model

OBJECTIVE The earliest response of esophageal mucosa to gastric reflux is the development of oxidative damage and inflammation. These processes contribute to the development of metaplasia known as Barretts esophagus, as well as the progression to malignancy. Secretory phospholipase A(2) is a mediator of inflammation with levels that are increased in Barretts metaplasia and carcinoma when compared with levels in normal samples. Our goal is to determine the role of secretory phospholipase A(2) in the development of reflux-associated changes in the esophageal mucosa. METHODS Secretory phospholipase A(2)-deficient mice (C57BL/6, n = 5) and mice known to express high levels of secretory phospholipase A(2) (BALB/c, n = 5) underwent side-to-side surgical anastomosis of the first portion of the duodenum and gastroesophageal junction, allowing exposure of esophageal mucosa to duodenal and gastric contents duodeno-gastroesophageal anastomosis. Control animals (n = 5) of each strain underwent laparotomy with esophagotomy and repair. Tissue was frozen in embedding medium. Hematoxylin and eosin staining and Ki67 and secretory phospholipase A(2) immunohistochemistry were used to evaluate esophageal tissue and its response to duodeno-gastroesophageal anastomosis. RESULTS Immunofluorescent staining confirmed the absence of secretory phospholipase A(2) in C57BL/6 mice and its presence in BALB/c mice. Hematoxylin and eosin staining demonstrated significant thickening of the esophageal mucosa in response to gastroesophageal reflux in the presence of secretory phospholipase A(2). Mice known to express high levels of secretory phospholipase A(2) also demonstrated increased numbers of proliferating cells. Secretory phospholipase A(2)-deficient mice were immune to the early changes induced by mixed reflux. CONCLUSIONS The presence of secretory phospholipase A(2) appears necessary for early histologic changes produced by exposure of the esophagus to gastroduodenal contents. This enzyme is identified as a promising target for evaluation of mechanisms of carcinogenesis and chemoprevention of esophageal carcinoma.

Hemoglobin-Based Oxygen Carrier Induces Heme Oxygenase-1 in the Heart and Lung but Not Brain

BACKGROUND The clinical sequel of ischemia and reperfusion remains a challenge in several clinical areas. Overexpression of heme oxygenase-1 (HO-1), using viral vectors, endotoxemia, and hypoxia, provides protection against ischemia and reperfusion injury. To date, however, no clinically viable therapy exists to safely induce HO-1. We have recently observed that administration of a hemoglobin-based oxygen carrier (HBOC) attenuates postinjury systemic inflammation. We have further demonstrated that an HBOC can induce HO-1 in vitro. We now explore the tissue-specific induction of heme oxygenase-1 after administration of an HBOC. STUDY DESIGN Rats were infused with doses of HBOC or saline through femoral vein injection (n=5 per group). Animals were sacrificed and organs were flushed. Heart, lung, and brain samples were taken for evaluation of total organ levels of HO-1 induction and for histologic localization of the cellular expression of the HO-1. Heat shock protein 72 levels were also analyzed to determine whether HO-1 induction was a generalized stress response. RESULTS Both the heart and lung demonstrated a dose-dependent induction of total organ HO-1. Interestingly, brain tissue did not have any significant amount of HO-1, either at baseline or after HBOC therapy. The cellular localization of HO-1 between organs was also specific, predominantly occurring in the cardiac myocyte and alveolar macrophages. Heat shock protein 72 levels were not significantly changed in any group examined, suggesting the induction of HO-1 is specific. CONCLUSIONS This study demonstrates that a clinically accessible product, HBOC, can specifically and selectively induce the expression of the protective enzyme HO-1 in vivo. These findings begin to characterize which organ systems may benefit by preischemic treatments with HBOC and further expand potential clinical applications of HBOCs.

A 31-Year-Old Woman With Hemoptysis and an Intrathoracic Mass

A 31-year-old woman presented with 3 months of hemoptysis (one-half cup daily), left chest pain, and intermittent expectoration. The sputum was described as having a “cheeselike” appearance, occurring every 5 to 6 days. She reported mild shortness of breath for 2 months but no weight loss, night sweats, or fevers. Her chest pain was episodic in nature and occasionally pleuritic. She denied nausea, vomiting, rashes, or joint pain. Her menses were regular and of normal quantity. Six years prior, she presented to another institution with an acute onset of hemoptysis and underwent left internal mammary arterial embolization. A diagnosis was not established, and she was lost to follow-up. The hemoptysis had subsided following the embolization but now returned. She underwent a left eye enucleation at age 4 years for retinoblastoma. She received no radiation, chemotherapy, or subsequent genetic evaluation. She reported no pneumonia, foreign-body aspiration, or coagulopathy. She was a former smoker (5 years), occasionally drank alcohol, and denied illicit drug use. She was from Denver and had not traveled outside of the United States. There were no risk factors for TB. She took no medications. There was no family history of retinoblastoma, other malignancies, or pulmonary disease. The physical examination included: temperature, 37.8°C; pulse, 64 beats/min; BP, 122/70 mm Hg; respiratory rate, 14 breaths/min; and oxygen saturation, 97% while breathing room air. She was thin and in no acute distress. There was a left prosthetic eye. An oropharyngeal examination revealed normal dentition and an absence of mucosal lesions. The neck was supple without masses or lesions. Cardiovascular exam revealed a normal S1, physiologically split S2, and no gallops, murmurs, or pericardial rub. Lung examination revealed decreased breath sounds over the anterior left thorax with early inspiratory crackles. The abdominal, lymphatic, and skin examinations were normal. Strength was normal. The WBC count was 10.0 × 103 cells/μL with 64% neutrophils, 24% lymphocytes, 6% monocytes, and 6% eosinophils; hemoglobin 14.1 g/dL; hematocrit 42.2%; and platelet count 296 × 103 cells/μL. The serum electrolytes, liver function tests, coagulation studies, and urinalysis were normal. HIV testing was negative. The chest radiograph is shown in Figures 1 and ​and2,2, and the CT scan is shown in Figure 3. Figure 1. Posteroanterior chest radiograph. Figure 2. Lateral chest radiograph. Figure 3. CT scan of the thorax. What Is the Differential Diagnosis? The patient has a large, solitary, parenchymal mass in the left hemithorax. The chest radiograph (Figs 1, ​,2)2) showed a heterogenous left paracardiac mass with central calcifications and embolization coils within the left internal mammary artery. A chest CT scan (Fig 2) showed a large 7 × 7 cm complex mass in the left anterior hemithorax containing fat, nonfat soft tissue, and osseous structures with partial lingular atelectasis. The thyroid gland appeared normal, and there were small bilateral axillary reactive lymph nodes. The presence of continued hemoptysis suggested a communication between the mass and an adjacent bronchus, and her transient improvement following embolization of the left internal mammary artery suggested altered vascular architecture. The possibilities include the following: (1) a benign or malignant parenchymal tumor, (2) a tumor located in the anterior mediastinum, (3) a fungal or mycobacterial infection, (4) a lung abscess, or (5) a hydatid cyst. The leading possibilities are an anterior mediastinal mass or a benign solitary parenchymal tumor. The absence of constitutional symptoms, lymphadenopathy, and further radiographic lesions argued against a diffuse or rapidly growing metastatic disease. The patient had no history of exposure to TB, and the mass involved the inferior and anterior aspect of the thorax, making TB less likely. A lung abscess of this size with bronchial communication more likely than not would result in fevers, purulent and foul-smelling sputum, and weight loss. In addition to the solid structure and marked heterogeneity of the computed tomographic image, there was no exposure history suggesting the possibility of a hydatid cyst. Hamartoma is the most common benign lung tumor, accounting for 75% of all benign lung nodules and 5% to 10% of solitary pulmonary nodules. Hamartomas most often present in the sixth or seventh decades1,2 but can occur in young adults as well. The diameter is usually < 6 cm,1,3 but they have been reported as large as 25.5 cm.4 The mass in this patient had a heterogenous appearance with a radiographically apparent focus of calcification. Calcification has been reported to occur in 3% to 32% of hamartoma cases but tends to have a punctate or “popcornlike” pattern.1,5,6 Calcification can also occur in other benign pulmonary tumors, including plasma cell granulomas and desmoid tumors. Plasma cell granulomas, also referred to as an inflammatory pseudotumor, most often present in childhood or adolescence and usually do not exceed 6 cm in transverse diameter.7 Desmoid tumors most often arise from the chest wall and invade surrounding structures, although they sometimes arise from the lung parenchyma.8 An intrapulmonary or mediastinal lipoma is unlikely to have this CT scan appearance because they tend to have uniform fatty attenuation. Additionally, intrapulmonary lipoma more often occurs on the right and in middle-aged men.9 Pulmonary leiomyoma would also have a homogenous radiographic appearance.10 Finally, in a young woman with recurrent, episodic hemoptysis, pulmonary endometrioma should be considered. Radiographically this presents as small pulmonary nodules and not a large heterogenous mass.11,12 It was difficult to determine based on the CT scan appearance whether this large complex mass originated from the anterior mediastinum or from the lung parenchyma. Thymoma, the most common anterior mediastinal mass, rarely has calcification, and if it occurs, the calcifications are small, curvilinear, or punctate.13 Invasive thymomas are rare, but sometimes they can extend into the lung causing hemoptysis.13 This is an unusual presentation of lymphoma because there are no systemic symptoms or diffuse lymphadenopathy. A mediastinal or intrapulmonary teratoma can present as a large, heterogenous mass with fat, soft tissue, and osseous structures, and can invade an adjacent bronchus. Occasionally patients can expectorate tissue from an intrabronchial tumor, which could account for this patient’s cheeselike sputum. What Should be Done Next? This patient had hemoptysis with recurrent symptoms. She was hemodynamically stable and did not have a coagulopathy. Although a tuberculin skin test should be performed and sputum could be examined for the presence of atypical cells or infection, the next logical step is to obtain tissue for a histologic diagnosis. Consideration should be given to performing a bronchoscopy to determine whether there is endobronchial involvement. Although surgical resection is not required for many hamartomas, the large size, bronchial invasion, and persistent symptoms caused by this mass represent indications for surgical resection. The sampling error of a percutaneous needle aspiration or transbronchial biopsy in such a heterogenous lesion is sufficiently high. Thus, surgical resection is recommended. What is the diagnosis? Diagnosis: mature intrapulm0onary teratoma The results of the workup revealed the following. Her tuberculin skin test was negative. At the time of surgery, a fiberoptic bronchoscopy was performed and did not reveal any abnormalities. Next, the patient was intubated with a double-lumen endotracheal tube, and a left lateral thoracotomy incision at the fifth intercostal space was undertaken. The large heterogenous mass appeared to originate from the lingula and was adherent to the mediastinal pleura and the anterolateral chest wall. During surgical manipulation of the mass, several hairs were noticed within the endotracheal tube. A complete lingulectomy was performed along with excision of an encapsulated 7×5.5×6.5-cm mass without complication. Pathologic evaluation revealed a thick, fibrous capsule with visible areas of calcification. The internal contents of the mass were separated into loculations by the fibrous capsule and included areas of a green-yellow granular substance with intermixed hair, soft adipose tissue, and a mucous white substance. Histologically, the resected specimen displayed a mature, intrapulmonary teratoma; it specifically showed well-differentiated tissues derived from all three germinal layers: respiratory, pancreatic, and intestinal epithelium from endoderm, pilosebaceous units from mesoderm, and hair follicles and squamous epithelium of skin from the ectoderm (Figs 4, ​,55). Figure 4. Hematoxylin-eosin-stained image from the resected intrathoracic mass demonstrating skin with hair follicle and sebaceous glands on the right. There is laminated keratin material on the left. Original magnification ×200. Figure 5. This hematoxylin-eosin-stained section demonstrates cartilage on the right and pancreatic acini on the left. Original magnification ×200.

Concomitant surgical cryoablation for refractory ventricular tachycardia and left ventricular assist device placement: a dual remedy but a recipe for thrombosis?

BackgroundVentricular tachycardia (VT) can persist following placement of a left ventricular assist device (LVAD). The optimal management strategy for VT during the peri-LVAD period is unknown.Case PresentationsTwo case reports are presented that describe epicardial and endocardial VT ablation performed during LVAD placement. Subsequently, both patients developed LVAD thrombosis, a known and dreaded complication of LVADs, requiring re-operation.ConclusionsWhile LVAD thrombosis is likely multifactorial and remains an area of active research, these two cases should increase awareness of the possible risks of VT ablation—especially endocardial ablation—during LVAD placement. Further research is needed to understand the effects of VT ablation during the peri-LVAD period.

Gastroduodenal reflux induces group IIa secretory phospholipase A2 expression and activity in murine esophagus

Exposure of esophageal epithelium to gastric and duodenal contents results in the histologic changes of hyperproliferation and mucosal thickening. We have previously shown that presence of secretory phospholipase A(2) (sPLA(2)) is necessary to produce these histologic changes in a murine model of gastroduodenal reflux. We sought to determine the influence of gastroduodenal reflux (GDR) on sPLA(2) protein and mRNA levels as well as enzyme activity in esophageal tissue. BALB/c (sPLA(2)(+/+)) mice (n= 28) underwent side-to-side surgical anastomosis of the first portion of the duodenum and GE junction (DGEA) resulting in continuous exposure of esophageal mucosa to mixed gastric and duodenal contents. Sham control mice (n= 14) underwent laparotomy, esophagotomy and closure. Real-time RT PCR was used to quantitate the influence of GDR on group IIa sPLA(2) expression. Immunofluorescent staining was quantitated by digital microscopy using a specific antibody to identify and locate sPLA(2) protein. A colorimetric assay was used to quantify total sPLA(2) activity after standardization of protein levels. Statistical analysis was conducted using Students t-test. Group IIa sPLA(2) mRNA and protein levels were increased at 4 and 8 weeks compared with sham controls. This increase occurred in a time-dependent manner and correlated with esophageal mucosal thickness. Furthermore, sPLA(2) enzyme activity was increased significantly at 4 and 8 weeks compared with untreated controls. The expression of group IIa sPLA(2) as well as sPLA(2) activity is induced by GDR. This novel finding indicates that sPLA(2) may play a role in the development of the histologic changes produced by GDR in esophageal mucosa.

Critical pathways leading to obliterative bronchiolitis in lung allografts

Purpose of reviewLung transplantation can prolong the life of patients with end-stage lung disease; however, long-term survival after transplantation is limited because of the fibrotic occlusion of small airways. This review aims to highlight recent developments in experimental/clinical research that attempt to explain how this process occurs. Recent findingsRecent investigations have focused on several pathways in the chronology of inflammation and fibrosis. Epithelial injury and resultant epithelial-mesenchymal transition have been demonstrated in other fibrotic diseases, and recent data suggest a similar role in the lung. Growth factors involved in the activation of fibroblasts to myofibroblasts and the promotion of collagen deposition appear to be a potent target to halt fibroproliferation. Studies suggest an important role for extracellular matrix remodeling, including angiogenesis, matrix metalloproteinases, and discoidin domain receptors. In addition, there is a growing body of literature regarding the innate immune response and injury as a result of gastroesophageal reflux disease. SummaryInflammation caused by an insult (alloimmune recognition, gastric reflux, etc.) appears to be the inciting factor in a cascade of events that lead to epithelial transition, growth factor production, myofibroblast activation, and extracellular matrix remodeling. An improved understanding of these critical pathways will facilitate the development of clinically useful therapies.

IP139. Management Recommendations for Arterial Entrapment of an Intra-Aortic Balloon Pump Caused by Balloon Rupture and Helium-Hemoglobin Clot

indicated care managers identified marginally more superficial wounds in the THEM group (31.3% vs 7.1%; P 1⁄4 .175). Both groups reported an increase in the 8-Item Short Form Health Survey physical summary scores, but it was more pronounced in THEM patients (P 1⁄4 .076). THEM patients reported a significantly greater improvement in quality of life on two of the 8-Item Short Form Health Survey quality subscales (roledphysical [THEM, 8.7; control, 1.1] and roleeemotional [THEM, 6.1; control, 0.5]; both P < .05). THEM patients reported trends for higher satisfaction in terms of general satisfaction, technical quality, and accessibility for Patient Satisfaction Questionnaire Short Form survey questions (4.2 vs 3.7 [P 1⁄4 .72], 4.5 vs 4.1 [P 1⁄4 .81], and 4.2 vs 3.8 [P 1⁄4 .63]), respectively. Conclusions: THEM was technically feasible and provided significant benefit to patients in geographically disparate areas. THEM was associated with increased patient satisfaction. Additional findings suggested that THEM patients embraced telehealth technology and took advantage of increased access to health care professionals. Telehealth successfully merged remotely generated information with care manager interaction. Presently, a larger study, preferably multicenter, is warranted and under consideration.