Rubin M. Tuder

Inhibition of VEGF receptors causes lung cell apoptosis and emphysema

Pulmonary emphysema, a significant global health problem, is characterized by a loss of alveolar structures. Because VEGF is a trophic factor required for the survival of endothelial cells and is abundantly expressed in the lung, we hypothesized that chronic blockade of VEGF receptors could induce alveolar cell apoptosis and emphysema. Chronic treatment of rats with the VEGF receptor blocker SU5416 led to enlargement of the air spaces, indicative of emphysema. The VEGF receptor inhibitor SU5416 induced alveolar septal cell apoptosis but did not inhibit lung cell proliferation. Viewed by angiography, SU5416-treated rat lungs showed a pruning of the pulmonary arterial tree, although we observed no lung infiltration by inflammatory cells or fibrosis. SU5416 treatment led to a decrease in lung expression of VEGF receptor 2 (VEGFR-2), phosphorylated VEGFR-2, and Akt-1 in the complex with VEGFR-2. Treatment with the caspase inhibitor Z-Asp-CH(2)-DCB prevented SU5416-induced septal cell apoptosis and emphysema development. These findings suggest that VEGF receptor signaling is required for maintenance of the alveolar structures and, further, that alveolar septal cell apoptosis contributes to the pathogenesis of emphysema.

Endothelial Dysfunction in Pulmonary Hypertension

The pathogenesis of pulmonary hypertension (PH) involves a complex and multifactorial process. Endothelial dysfunction seems to play an integral role in mediating the structural changes in the pulmonary vasculature. Disordered endothelial cell proliferation along with concurrent neoangiogenesis, when exuberant, results in the formation of glomeruloid structures known as the plexiform lesions, which are common pathological features of the pulmonary vessels of patients with pulmonary arterial hypertension (PAH). In addition, an altered production of various endothelial vasoactive mediators, such as NO, prostacyclin, endothelin-1 (ET-1), serotonin, and thromboxane, has been increasingly recognized in patients with PH. Because most of these mediators affect the growth of the smooth muscle cells, an alteration in their production may facilitate the development of pulmonary vascular hypertrophy and structural remodeling characteristic of PH. It is conceivable that the beneficial effects of many of the treatments currently available for PAH, such as the use of prostacyclin, NO, and ET antagonists, result at least in part from restoring the balance between these mediators. However, the ultimate cellular and physiological targets of these treatments remain unknown. In addition to the potential consequences of an imbalance in the endothelial production of various mediators, injury to the endothelium may expose the underlying vascular tissue to diverse blood-borne factors that may further promote pathological changes. Endothelial dysfunction may also have adverse consequences on pulmonary vascular hemostasis by altering the production of anticoagulant factors. Recent reports of genetic mutations in the endothelial cells of patients with PH further underscore the role of these cells in the disease pathogenesis. The endothelium lining the normal lung is characterized by significant heterogeneity. Not only is it vastly different from systemic endothelium in ultrastructure and function, but it varies in various vessel types in the pulmonary vasculature itself.1 The main functions of the pulmonary endothelium include maintenance …

Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension

Our understanding of the pathobiology of severe pulmonary hypertension, usually a fatal disease, has been hampered by the lack of information of its natural history. We have demonstrated that, in human severe pulmonary hypertension, the precapillary pulmonary arteries show occlusion by proliferated endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR‐2) are involved in proper maintenance, differentiation, and function of endothelial cells. We demonstrate here that VEGFR‐2 blockade with SU5416 in combination with chronic hypobaric hypoxia causes severe pulmonary hypertension associated with precapillary arterial occlusion by proliferating endothelial cells. Prior to and concomitant with the development of severe pulmonary hypertension, lungs of chronically hypoxic SU5416‐treated rats show significant pulmonary endothelial cell death, as demonstrated by activated caspase 3 immunostaining and TUNEL. The broad caspase inhibitor Z‐Asp‐CH2DCB prevents the development of intravascular pulmonary endothelial cell growth and severe pulmonary hypertension caused by the combination of SU5416 and chronic hypoxia.—Taraseviciene‐Stewart, L., Kasahara, Y., Alger, L., Hirth, P., McMahon, G., Waltenberger, J., Voelkel, N. F., Tuder, R. M. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death‐dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J. 15, 427‐438 (2001)

Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice

Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma.

Plasma and lipids from stored packed red blood cells cause acute lung injury in an animal model.

Transfusion-related acute lung injury (TRALI) is a serious complication of hemotherapy. During blood storage, lipids are generated and released into the plasma. In this study, the role of these lipids in TRALI was investigated using an isolated, perfused rat lung model. Rats were pretreated with endotoxin (LPS) or saline in vivo and the lungs were isolated, ventilated, and perfused with saline, or (a) 5% (vol/ vol) fresh human plasma, (b) plasma from stored blood from the day of isolation (D.0) or from the day of outdate (D.42), (c) lipid extracts from D.42 plasma, or (d) purified lysophosphatidylcholines. Lungs from saline or LPS-pretreated rats perfused with fresh (D.0) plasma showed no pulmonary damage as compared with saline perfused controls. LPS pretreatment/D.42 plasma perfusion caused acute lung injury (ALI) manifested by dramatic changes in both pulmonary artery pressure and edema. Incubation of LPS pre-tx rats with mibefradil, a Ca2+ channel blocker, or WEB 2170, a platelet-activating factor (PAF) receptor antagonist, inhibited ALI caused by D.42 plasma. Lung histology showed neutrophil sequestration without ALI with LPS pretreatment/saline or D.0 plasma perfusion, but ALI with LPS pretreatment/D.42 plasma perfusion, and inhibition of D.42 plasma induced ALI with WEB 2170 or mibefradil. A significant increase in leukotriene E4 was present in LPS-pretreated/D.42 plasma-perfused lungs that was inhibited by WEB 2170. Lastly, significant pulmonary edema was produced when lipid extracts of D.42 plasma or lysophosphatidylcholines were perfused into LPS-pretreated lungs. Lipids caused ALI without vasoconstriction, except at the highest dose employed. In conclusion, both plasma and lipids from stored blood produced pulmonary damage in a model of acute lung injury. TRALI, like the adult respiratory distress syndrome, may be the result of two insults: one derived from stored blood and the other from the clinical condition of the patient.

Short telomeres are a risk factor for idiopathic pulmonary fibrosis

Idiopathic interstitial pneumonias (IIPs) have a progressive and often fatal course, and their enigmatic etiology has complicated approaches to effective therapies. Idiopathic pulmonary fibrosis (IPF) is the most common of IIPs and shares with IIPs an increased incidence with age and unexplained scarring in the lung. Short telomeres limit tissue renewal capacity in the lung and germ-line mutations in telomerase components, hTERT and hTR, underlie inheritance in a subset of families with IPF. To examine the hypothesis that short telomeres contribute to disease risk in sporadic IIPs, we recruited patients who have no family history and examined telomere length in leukocytes and in alveolar cells. To screen for mutations, we sequenced hTERT and hTR. We also reviewed the cases for features of a telomere syndrome. IIP patients had shorter leukocyte telomeres than age-matched controls (P < 0.0001). In a subset (10%), IIP patients had telomere lengths below the first percentile for their age. Similar to familial cases with mutations, IPF patients had short telomeres in alveolar epithelial cells (P < 0.0001). Although telomerase mutations were rare, detected in 1 of 100 patients, we identified a cluster of individuals (3%) with IPF and cryptogenic liver cirrhosis, another feature of a telomere syndrome. Short telomeres are thus a signature in IIPs and likely play a role in their age-related onset. The clustering of cryptogenic liver cirrhosis with IPF suggests that the telomere shortening we identify has consequences and can contribute to what appears clinically as idiopathic progressive organ failure in the lung and the liver.

MONOCLONAL ENDOTHELIAL CELL PROLIFERATION IS PRESENT IN PRIMARY BUT NOT SECONDARY PULMONARY HYPERTENSION

The etiology and pathogenesis of the vascular lesions characterizing primary pulmonary hypertension (PPH), an often fatal pulmonary vascular disease, are largely unknown. Plexiform lesions composed of proliferating endothelial cells occur in between 20 and 80% of the cases of this irreversible pulmonary vascular disease. Recently, technology to assess monoclonality has allowed the distinction between cellular proliferation present in neoplasms from that in reactive nonneoplastic tissue. To determine whether the endothelial cell proliferation in plexiform lesions in PPH is monoclonal or polyclonal, we assessed the methylation pattern of the human androgen receptor gene by PCR (HUMARA) in proliferated endothelial cells in plexiform lesions from female PPH patients (n = 4) compared with secondary pulmonary hypertension (PH) patients (n = 4). In PPH, 17 of 22 lesions (77%) were monoclonal. However, in secondary PH, all 19 lesions examined were polyclonal. Smooth muscle cell hyperplasia in pulmonary vessels (n = 11) in PPH and secondary PH was polyclonal in all but one of the examined vessels. The monoclonal expansion of endothelial cells provides the first marker that allows the distinction between primary and secondary PH. Our data of a frequent monoclonal endothelial cell proliferation in PPH suggests that a somatic genetic alteration similar to that present in neoplastic processes may be responsible for the pathogenesis of PPH.

Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice

Alveolar cell apoptosis is involved in the pathogenesis of emphysema, a prevalent disease primarily caused by cigarette smoking. We report that ceramide, a second messenger lipid, is a crucial mediator of alveolar destruction in emphysema. Inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis, oxidative stress and emphysema caused by blockade of the vascular endothelial growth factor (VEGF) receptors in both rats and mice. Emphysema was reproduced with intratracheal instillation of ceramide in naive mice. Excessive ceramide triggers a feed-forward mechanism mediated by activation of secretory acid sphingomyelinase, as suggested by experiments with neutralizing ceramide antibody in mice and with acid sphingomyelinase–deficient fibroblasts. Concomitant augmentation of signaling initiated by a prosurvival metabolite, sphingosine-1-phosphate, prevented lung apoptosis, implying that a balance between ceramide and sphingosine-1-phosphate is required for maintenance of alveolar septal integrity. Finally, increased lung ceramides in individuals with smoking-induced emphysema suggests that ceramide upregulation may be a crucial pathogenic element and a promising target in this disease that currently lacks effective therapies.

Expression of angiogenesis-related molecules in plexiform lesions in severe pulmonary hypertension: evidence for a process of disordered angiogenesis.

Pulmonary arteries of patients with severe pulmonary hypertension (SPH) presenting in an idiopathic form (primary PH‐PPH) or associated with congenital heart malformations or collagen vascular diseases show plexiform lesions. It is postulated that in lungs with SPH, endothelial cells in plexiform lesions express genes encoding for proteins involved in angiogenesis, in particular, vascular endothelial growth factor (VEGF) and those involved in VEGF receptor‐2 (VEGFR‐2) signalling. On immunohistochemistry and in situ hybridization, endothelial cells in the plexiform lesions expressed VEGF mRNA and protein and overexpressed the mRNA and protein of VEGFR‐2, and the transcription factor subunits HIF‐1α and HIF‐1β of hypoxia inducible factor, which are responsible for the hypoxia‐dependent induction of VEGF. When compared with normal lungs, SPH lungs showed decreased expression of the kinases PI3 kinase and src, which, together with Akt, relay the signal transduction downstream of VEGFR‐2. Because markers of angiogenesis are expressed in plexiform lesions in SPH, it is proposed that these lesions may form by a process of disordered angiogenesis. Copyright

DIFFUSE ALVEOLAR HEMORRHAGE AND SYSTEMIC LUPUS ERYTHEMATOSUS : CLINICAL PRESENTATION, HISTOLOGY, SURVIVAL, AND OUTCOME

Diffuse alveolar hemorrhage (DAH) complicating systemic lupus erythematosus (SLE) remains a devastating pulmonary complication of this systemic disease. We conducted this study to review the clinicopathologic presentation and the effects of prior treatment, presence of infection, and current treatment on the survival and outcome of patients with DAH and SLE. We reviewed the records of 15 SLE patients who experienced 19 episodes of DAH over a 10-year period in a single tertiary care hospital. These patients were compared with 57 previously reported cases. The 19 episodes of DAH represented 3.7% of the 510 admissions occasioned by various complications of SLE. As previously reported, the majority (66%) were women with a median age of 27 years. The onset was often abrupt: < 3 days in 12 of the episodes. In 3 patients (20%), DAH was the initial manifestation of SLE, compared with 11% in the literature series. In the other patients in the present series, DAH appeared a median of 31 months following the diagnosis of SLE, versus 35 months in the literature series. In only 42% of the episodes in the present series, compared with 66% in the literature series, was hemoptysis present at the time of admission. However, hemoptysis eventually appeared in all 19 episodes. Temperature elevation (> 38 degrees C) was another inconsistent finding, found in only 5 episodes (26%) in the present series. The most constant concurrent systemic finding was lupus nephritis (14/15 patients). This represents a significant increase when compared with the literature series (29/48 patients). In 8 of 10 patients in whom lung tissue was available, pulmonary capillaritis accompanied the DAH. This represents a marked difference in the underlying histologic pattern when compared with the literature series. In those patients, 72% (31/43 patients) had bland pulmonary hemorrhage, and capillaritis was described in only 6 patients. The overall patient mortality rate was 53% in the current series and 50% in the literature series. Factors associated with an increased mortality in the present series include the following: mechanical ventilation (62%) versus no mechanical ventilation (0%); infection (78%) versus no infection (20%); and cyclophosphamide therapy for the acute DAH episode (70%) versus no cyclophosphamide therapy (20%). The incidence of infection in DAH and SLE (9/19 episodes) is far greater than previously reported (7/ 57 episodes). One possible explanation for this difference is the increased use of outpatient immunosuppressive therapy with monthly intravenous cyclophosphamide therapy for lupus nephritis. Eighteen DAH episodes in the present series were treated with intravenous methylprednisolone. When one combines both the current and literature series experience (16 episodes), the use of plasmapheresis does not improve survival. Of the 7 patients in the present series who survived all episodes of DAH, 6 remain alive a median of 50 months post episode and without recurrence of DAH. Diffuse alveolar hemorrhage is an uncommon but lethal complication of SLE. The survival rate remains unchanged from previous reports. The absence of hemoptysis should not exclude this diagnosis, particularly in those patients who experience an acute pulmonary syndrome with new radiographic infiltrates accompanied by falling hematocrit and the presence of a hemorrhagic bronchoalveolar lavage. Evidence for lupus nephritis is present in the great majority of cases. Most cases demonstrate the histologic pattern of pulmonary capillaritis. The mortality is adversely affected by the need for mechanical ventilation, either the presence of infection at the time of admission or the development of infection in the hospital, and the use of cyclophosphamide for treatment of the acute event.