We have previously demonstrated that a failure of pulmonary endothelial cell survival induced by vascular endothelial growth factor (VEGF) receptor blockade results in lung alveolar septal cell apoptosis and emphysema. Because apoptosis and oxidative stress may be pathobiologically linked, we hypothesized that oxidative stress has a central role in alveolar septal cell apoptosis and emphysema induced by VEGF receptor blockade. When compared with control animals, rats treated with the VEGF receptor blocker SU5416 showed increased alveolar enlargement, alveolar septal cell apoptosis, and expression of markers of oxidative stress, all of which were prevented by the superoxide dismutase mimetic M40419. The preservation of lung structure in SU5416+M40419-treated lungs was associated with increased septal cell proliferation, and enhanced phosphorylation of the prosurvival and antiapoptotic Akt, when compared with SU5416-treated lungs. Consistent with a positive feedback interaction between oxidative stress and apoptosis, we found that apoptosis predominated in areas of oxidative stress, and that apoptosis blockade by a broad spectrum caspase inhibitor markedly reduced the expression of markers of oxidative stress induced by SU5416 treatment. Oxidative stress and apoptosis, which cause lung cellular destruction in emphysema induced by VEGF receptor blockade, may be important mediators common to human and experimental emphysema.

Removal of apoptotic cells by neighboring viable cells or professional phagocytes is essential for the maintenance of tissue homeostastis. Here we show that the phagocytosis of apoptotic Jurkat T cells by mouse epithelial cells (HC-11) and peritoneal macrophages leads to the secretion of growth and survival factors. We characterized VEGF as one of these factors which subsequently promote the proliferation of endothelial cells. Further we demonstrate that the phagocytosis of apoptotic bodies inhibits both spontanous and UV-irradiation-induced apoptosis in endothelial and epithelial cells. These effects were not observed when phagocytes had been exposed to viable or necrotic Jurkat T cells. We conclude that phagocytosis of apoptotic cells leads to secretion of growth and survival factors by phagocytes that represents a new form of life-promoting cell-cell interaction.

In spite of treatment, severe angioproliferative pulmonary arterial hypertension (PAH) remains a disease characterized by great morbidity and shortened survival. New treatment strategies for patients with PAH are needed, and after drug development, preclinical studies are best conducted in animal models which present with pulmonary angio-obliterative disease and right heart failure. A rat model of severe pulmonary hypertension and right heart failure, described a decade ago, continues to be investigated and provide insight into the nature of the lung vascular lesions and mechanisms of cardiac adaptation to an altered lung circulation. This rat model is based on the combination of VEGF receptor blockade with Su5416 and chronic hypoxia; use of this pulmonary hypertension induction strategy led to developing the concept of apoptosis-dependent compensatory vascular cell growth. Although, often employed in experimental designs, chronic hypoxia is not necessary for the development of angio-obliterative pulmonary hypertension. Left pneumonectomy combined with Su5416 also results in severe pulmonary hypertension in normoxic conditions. Similarly, the immune insufficiency component of severe PAH can be modeled in athymic rats (lacking T-lymphocytes). In these rats housed under normoxic conditions, treatment with the VEGFR receptor blocker results in angioproliferative pulmonary hypertension; cardiopulmonary disease in these animals can be prevented by immune reconstitution of regulatory T-cells (Tregs). Finally, chronic hypoxia can be replaced with another stimulator of HIF-1α: Ovalbumin (Ova). Immunization of rats with Ova increases lung tissue HIF-1α protein expression, and in Su5416-treated rats causes lethal pulmonary hypertension. Finally, we postulate that these models may also be useful for “reverse translation”; that is, the mechanisms of lung vascular cell death and growth and the modifying influences of immune and bone marrow cells that have been identified in the Su5416 VEGFR inhibitor models can be informative about heretofore undescribed processes in human PAH.

IL-32 is a multi-faceted cytokine with a role in infections, autoimmune diseases, and cancer, and it exerts diverse functions, including aggravation of inflammation and inhibition of virus propagation. We previously identified IL-32 as a critical regulator of endothelial cell (EC) functions, and now reveal that IL-32 also possesses angiogenic properties. The hyperproliferative EC of human pulmonary arterial hypertension (PAH) and glioblastoma multiforme exhibited a markedly increased abundance of IL-32, and, significantly, the cytokine colocalized with integrin αVβ3. VEGF receptor blockade, which resulted in EC hyperproliferation, increased IL-32 threefold. siRNA-mediated silencing of IL-32 negated the 58% proliferation of EC that occurred within 24h in scrambled-transfected controls. Reduction of IL-32 neither affected apoptosis (insignificant changes in Bak-1, Bcl-2, Bcl-XL, LDH, annexin V, and propidium iodide) nor VEGF or TGF-β levels, but siIL-32-transfected adult and neonatal EC produced up to 61% less NO, IL-8, and MMP-9, and up to 3-fold more activin A and endostatin. In co-culture-based angiogenesis assays, IL-32γ dose-dependently increased tube formation up to 3-fold; an αVβ3 inhibitor prevented this activity, and reduced IL-32γ-induced IL-8 by 85%. In matrigel plugs loaded with IL-32γ, VEGF, or vehicle, and injected into live mice, we observed the anticipated VEGF-induced increase in neocapillarization (8-fold vs vehicle), but unexpectedly, IL-32γ was equally angiogenic. A second signal such as IFNγ was required to render cells responsive to exogenous IL-32γ; importantly, this was confirmed using a completely synthetic preparation of IL-32γ. In summary, we add angiogenic properties that are mediated by integrin αVβ3 but VEGF-independent, to the portfolio of IL-32, implicating a role for this versatile cytokine in PAH and neoplastic diseases.

The alveolar destruction leading to airspace enlargement in patients with end-stage chronic obstructive pulmonary disease (COPD) is frequently progressive, despite smoking cessation. Several laboratories have accumulated data demonstrating the presence of immune cells in bronchial biopsy specimens and lung tissue sections from patients with COPD. Recently, the accumulation of T and B lymphocytes, often forming follicles, in the lung parenchyma from patients with severe COPD has been reported. In addition, it has been postulated that there might be an autoimmune component to COPD. T-cell receptor analysis has provided data consistent with the concept of T-cell clones in the lung tissue from patients with COPD. Against this background, we developed a model of autoimmune emphysema in adult rats. Based on published data showing that immunization of mice with human umbilical vein endothelial cells (HUVECs) causes production of anti-vascular endothelial growth factor (VEGF) receptor II (KDR) antibodies, and our own data indicating that administration of a VEGF receptor blocker in adult rats causes emphysema, we reasoned that intraperitoneal injection of HUVECs in rats would generate both anti-VEGF receptor antibodies and emphysema. Indeed, intraperitoneal injection of HUVECs caused emphysema. We further explored the autoimmune nature of this model, identified KDR antibodies in the serum of HUVEC-immunized rats, and injected serum from the emphysematous rats into naive rats and mice, which resulted in emphysema. Presently, we are in the process of investigating whether cigarette smoke extract causes emphysema. We recently identified anti-endothelial cell antibodies in the serum of patients with end-stage emphysema.