Chronic Embolic Pulmonary Hypertension Caused by Pulmonary Embolism and Vascular Endothelial Growth Factor Inhibition.

Our understanding of the pathophysiological basis of chronic thromboembolic pulmonary hypertension (CTEPH) will be accelerated by an animal model that replicates the phenotype of human CTEPH. Sprague-Dawley rats were administered a combination of a single dose each of plastic microspheres and vascular endothelial growth factor receptor antagonist in polystyrene microspheres (PE) + tyrosine kinase inhibitor SU5416 (SU) group. Shams received volume-matched saline; PE and SU groups received only microspheres or SU5416, respectively. PE + SU rats exhibited sustained pulmonary hypertension (62 ± 13 and 53 ± 14 mmHg at 3 and 6 weeks, respectively) with reduction of the ventriculoarterial coupling in vivo coincident with a large decrement in peak rate of oxygen consumption during aerobic exercise, respectively. PE + SU produced right ventricular hypokinesis, dilation, and hypertrophy observed on echocardiography, and 40% reduction in right ventricular contractile function in isolated perfused hearts. High-resolution computed tomographic pulmonary angiography and Ki-67 immunohistochemistry revealed abundant lung neovascularization and cellular proliferation in PE that was distinctly absent in the PE + SU group. We present a novel rodent model to reproduce much of the known phenotype of CTEPH, including the pivotal pathophysiological role of impaired vascular endothelial growth factor-dependent vascular remodeling. This model may reveal a better pathophysiological understanding of how PE transitions to CTEPH in human treatments.

Structural and functional characterization of endothelial microparticles released by cigarette smoke.

Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.

Topical application of a platelet activating factor receptor agonist suppresses phorbol ester-induced acute and chronic inflammation and has cancer chemopreventive activity in mouse skin.

Platelet activating factor (PAF) has long been associated with acute edema and inflammatory responses. PAF acts by binding to a specific G-protein coupled receptor (PAF-R, Ptafr). However, the role of chronic PAF-R activation on sustained inflammatory responses has been largely ignored. We recently demonstrated that mice lacking the PAF-R (Ptafr-/- mice) exhibit increased cutaneous tumorigenesis in response to a two-stage chemical carcinogenesis protocol. Ptafr-/- mice also exhibited increased chronic inflammation in response to phorbol ester application. In this present study, we demonstrate that topical application of the non-hydrolysable PAF mimetic (carbamoyl-PAF (CPAF)), exerts a potent, dose-dependent, and short-lived edema response in WT mice, but not Ptafr -/- mice or mice deficient in c-Kit (c-KitW-sh/W-sh mice). Using an ear inflammation model, co-administration of topical CPAF treatment resulted in a paradoxical decrease in both acute ear thickness changes associated with a single PMA application, as well as the sustained inflammation associated with chronic repetitive PMA applications. Moreover, mice treated topically with CPAF also exhibited a significant reduction in chemical carcinogenesis. The ability of CPAF to suppress acute and chronic inflammatory changes in response to PMA application(s) was PAF-R dependent, as CPAF had no effect on basal or PMA-induced inflammation in Ptafr-/- mice. Moreover, c-Kit appears to be necessary for the anti-inflammatory effects of CPAF, as CPAF had no observable effect in c-KitW-sh/W-sh mice. These data provide additional evidence that PAF-R activation exerts complex immunomodulatory effects in a model of chronic inflammation that is relevant to neoplastic development.

Mice lacking epidermal PPARγ exhibit a marked augmentation in photocarcinogenesis associated with increased UVB-induced apoptosis, inflammation and barrier dysfunction.

Recent studies suggest that peroxisome proliferator-activated receptor gamma (PPARγ) agonists may have cancer chemopreventive activity. Other studies have shown that loss of epidermal PPARγ results in enhanced chemical carcinogenesis in mice via unknown mechanisms. However, ultraviolet B (UVB) exposure represents the primary etiological agent for skin cancer formation and the role of PPARγ in photobiology and photocarcinogenesis is unknown. In previous studies, we demonstrated that UVB irradiation of cells results in the formation of oxidized glycerophosphocholines that exhibit PPARγ ligand activity. We therefore hypothesized that PPARγ would prove to be a chemopreventive target in photocarcinogenesis. We first showed that UVB irradiation of mouse skin causes generation of PPARγ agonist species in vivo. We then generated SKH-1 hairless, albino mice deficient in epidermal Pparg (Pparg-/-(epi)) using a cytokeratin 14 driven Cre-LoxP strategy. Using a chronic model of UVB photocarcinogenesis, we next showed that Pparg-/-(epi) mice exhibit an earlier onset of tumor formation, increased tumor burden and tumor progression. Increased tumor burden in Pparg-/-(epi) mice was accompanied by a significant increase in epidermal hyperplasia and p53 positive epidermal cells in surrounding skin lacking tumors. After acute UVB irradiation, Pparg-/-(epi) mice exhibited an augmentation of both UVB-induced Caspase 3/7 activity and inflammation. Increased apoptosis and inflammation was also observed after treatment with the PPARγ antagonist GW9662. With chronic UVB irradiation, Pparg-/-(epi) mice exhibited a sustained increase in erythema and transepidermal water loss relative to wildtype littermates. This suggests that PPARγ agonists could have possible chemopreventive activity in non-melanoma skin cancer.

Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity.

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.

Loss of the platelet activating factor receptor in mice augments PMA-induced inflammation and cutaneous chemical carcinogenesis.

Although platelet-activating factor (PAF) is a well-known acute inflammatory mediator, little is known regarding the role of PAF in chronic inflammation. Phorbol esters are known to stimulate PAF production. Moreover, the ability of repeated applications of phorbol esters to induce a sustained inflammatory response is crucial to their tumorigenic activity. We therefore examined whether PAF acts as a mediator of phorbol ester-induced inflammation and tumorigenesis. While PAF receptor knockout mice (PAFR(-/-)) showed an expected but modest reduction in the acute inflammatory response to phorbol 12-myristate 13-acetate (PMA), these mice exhibited a surprising increase in inflammation following chronic PMA application. This increased inflammation was documented by a number of findings that included: increased skin thickness, increased myeloperoxidase activity and expression and increased expression of known inflammatory mediators. Interestingly, vehicle-treated PAFR(-/-) mice also exhibited modest increases in levels of inflammatory markers. This suggests that the platelet activating factor receptor (PAFR) acts to suppress chronic inflammation in response to other stimuli, such as barrier disruption. The idea that chronic PAFR activation is anti-inflammatory was documented by repetitive topical PAFR agonist administration that resulted in reduced myeloperoxidase activity in skin. We next utilized a 7,12-dimethylbenz(a)anthracene/PMA carcinogenesis protocol to demonstrate that PAFR(-/-) mice exhibit significantly increased tumor formation and malignant progression compared with wild-type control mice. These studies provide evidence for two important, unexpected and possibly interrelated pathological roles for the PAFR: first, the PAFR acts to suppress PMA-induced chronic inflammation; secondly, the PAFR acts to suppress neoplastic development in response to chemical carcinogens.