Microparticles Carrying Sonic Hedgehog Are Increased in Humans with Peripheral Artery Disease.

Sonic hedgehog (Shh) is a prototypical angiogenic agent with a crucial role in the regulation of angiogenesis. Experimental studies have shown that Shh is upregulated in response to ischemia. Also, Shh may be found on the surface of circulating microparticles (MPs) and MPs bearing Shh (Shh + MPs) have shown the ability to contribute to reparative neovascularization after ischemic injury in mice. The goal of this study was to test the hypothesis that, in humans with peripheral artery disease (PAD), there is increased number of circulating Shh + MPs. This was done by assessing the number of Shh + MPs in plasma of patients with PAD and control subjects without PAD. We found significantly higher number of Shh + MPs in plasma of subjects with PAD, compared to controls, while the global number of MPs-produced either by endothelial cells, platelets, leukocytes, and erythrocytes-was not different between PAD patients and controls. We also found a significant association between the number of Shh + MPs and the number of collateral vessels in the ischemic limbs of PAD patients. Interestingly, the concentration of Shh protein unbound to MPs-which was measured in MP-depleted plasma-was not different between subjects with PAD and the controls, indicating that, in the setting of PAD, the call for Shh recapitulation does not lead to secretion of protein into the blood but to binding of the protein to the membrane of MPs. These findings provide novel information on Shh signaling during ischemia in humans, with potentially important biological and clinical implications.

Microparticles Produced by Activated Platelets Carry a Potent and Functionally Active Angiogenic Signal in Subjects with Crohn's Disease.

Microparticles (MPs) are submicron vesicles shed from various cell types upon activation, stimulation, and death. Activated platelets are an important source of circulating MPs in subjects with inflammatory diseases, including Crohn's disease (CD). Angiogenesis is a hallmark of inflammation in CD and plays an active role in sustaining disease progression, while targeting angiogenesis may be an effective approach to block colitis. In this study, we analyzed the angiogenic content of the MPs produced by activated platelets in subjects with CD. We also evaluated whether the angiogenic signal carried by these MPs was functionally active, or able to induce angiogenesis. We found that, in subjects with CD, MPs produced by activated platelets contain significantly higher levels of angiogenic mRNAs, such as epidermal growth factor (EGF), platelet-derived growth factor-α (PDGFα), fibroblast growth factor (FGF-2), and angiopoietin-1 (ANGPT1), compared to MPs isolated from control subjects. They also contain significantly higher levels of prototypical angiogenic proteins, including vascular endothelial growth factor (VEGF), angiopoietin-1, endoglin, endothelin-1, pentraxin 3, platelet factor-4, plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinases-1 (TIMP-1), and thrombospondin 1. The protein content of these MPs is functionally active, since it has the ability to induce a robust angiogenic process in an endothelial cell/interstitial cell co-culture in vitro assay. Our results reveal a potential novel mechanism through which the angiogenic signal is delivered in subjects with CD, with potentially important clinical and therapeutic implications.

HepPar1-Positive Circulating Microparticles Are Increased in Subjects with Hepatocellular Carcinoma and Predict Early Recurrence after Liver Resection.

Circulating microparticles (MPs) are novel potential biomarkers in cancer patients. Their role in hepatocellular carcinoma (HCC) is under intensive investigation. In this study, we tested the hypothesis that MPs expressing the antigen HepPar1 are increased in the blood of subjects with HCC and may serve as markers of early recurrence after liver resection (LR). We studied 15 patients affected by HCC undergoing LR, and used flow cytometry to assess the number of circulating HepPar1+ MPs. Ten subjects without HCC (five with liver cirrhosis and five with healthy livers) were used as controls. After LR, HCC patients underwent a follow-up to check for early recurrence, which occurred in seven cases. The number of circulating HepPar1+ MPs was significantly higher in subjects affected by HCC, compared to individuals without cancer (p < 0.01). We also found that, among HCC patients, the number of circulating HepPar1+ MPs, measured before LR, was significantly higher in those who displayed early recurrence compared to those without recurrence (p = 0.02). Of note, other types of circulating MPs, such as those derived from endothelial cells (CD144+) or those produced by the activated endothelium (CD144+/CD62+), were not associated with HCC, nor could they predict HCC recurrence. HepPar1+ MPs deserve further investigation as novel biomarkers of disease and prognosis in HCC patients.

Angiogenic impairment of the vascular endothelium: a novel mechanism and potential therapeutic target in muscular dystrophy.

OBJECTIVE: Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs. APPROACH AND RESULTS: We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx-derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated ß-galactosidase activity. Mdx-derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell-derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density. CONCLUSIONS: These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy.