Effect of positive airway pressure therapy of obstructive sleep apnea on circulating Angiopoietin-2.

BACKGROUND: Obstructive sleep apnea (OSA) has been identified as a possible contributor to interstitial lung disease. While positive airway pressure (PAP) is effective therapy for OSA, it causes large increases in lung volumes during the night that are potentially deleterious, analogous to ventilator-induced lung injury, although this has not been previously studied. The goal of this study was to assess the impact of PAP therapy on four biomarkers of alveolar epithelial and endothelial injury and extracellular matrix remodeling in patients with OSA. METHODS: In 82 patients with moderate to severe OSA who were adherent to PAP therapy, surfactant protein D, osteopontin, angiopoietin-2, and matrix metalloprotease-7 were measured by ELISA in serum samples collected before and 3- to 6-months after initiation of PAP therapy. RESULTS: An increase in angiopoietin-2 level of 0.28 ng/mL following PAP therapy was observed (p = 0.007). This finding was replicated in an independent sample of OSA patients. No significant change was detected in surfactant protein D, osteopontin, or matrix metalloprotease-7. CONCLUSIONS: This finding raises concern for a possible adverse impact of PAP therapy on vascular endothelium.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Accelerate Cutaneous Wound Healing by Enhancing Angiogenesis through Delivering Angiopoietin-2.

The underlying mechanisms of human umbilical cord mesenchymal stem cells (hucMSCs) and their exosomes (hucMSC-Exs), which play significant roles in skin wound healing, remain poorly understood. By using a rat model of deep second-degree burn injury, the roles of hucMSC-Exs in angiogenesis and cutaneous wound healing in vivo were investigated. We found that hucMSC-Exs accelerated skin wound healing and angiogenesis, inducing a higher wound-closure rate and increased expression of CD31 in vivo. We also discovered that hucMSC-Exs contained angiopoietin-2 (Ang-2), and treatment with hucMSC-Exs enhanced the expression of the Ang-2 protein in the wound area and human umbilical vein endothelial cells (HUVECs) through exosomal-mediated Ang-2 transfer. Moreover, hucMSC-Exs promoted the proliferative, migratory, and tube-forming ability of HUVECs. Furthermore, overexpression of Ang-2 in hucMSC-Exs further enhanced HUVEC migration and tube formation and exerted therapeutic and proangiogenic effects in cutaneous wounds in rats, whereas knockdown of Ang-2 in hucMSC-Exs abrogated these therapeutic and proangiogenic effects. Taken together, our results indicated that hucMSC-Ex-derived Ang-2 plays a significant role in tube formation of HUVECs and promotion of angiogenesis, and further suggested that hucMSC-Ex-based therapy may serve as a promising therapeutic approach for promoting cutaneous wound healing.

Angiopoietin 2 promotes angiogenesis in tissue-engineered bone and improves repair of bone defects by inducing autophagy.

Angiogenesis plays a key role in the repair of large segmental bone defects with tissue-engineered bones. However, there is no effective method of promoting angiogenesis in tissue-engineered bone. Both angiopoietin 2 (Ang2) and autophagy have been shown to be involved in angiogenesis, but their roles in angiogenesis of tissue-engineered bone remains unknown. In this in vivo study, a radius bone defect was created in New Zealand white rabbits, which were then treated by implantation of a hydroxyapatite/collagen scaffold followed by injection of different concentrations of Ang2. Expression of the autophagic modulators microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, and SQSTM1/P62 were measured via western blotting, while the angiogenic modulators VEGF and CD31 were detected by western blotting and immunohistochemistry, respectively. X-ray imaging combined with general observation was used to evaluate bone defect healing. Expression of LC3 -I/LC3-II, Beclin-1, VEGF, and CD31 in the callus area increased and SQSTM1/p62 decreased in a dose-dependent manner with increasing Ang2 concentration. In the group treated with a high concentration of Ang2, the new callus grew well, accompanied by remarkable angiogenesis, leading to good repair of the bone defects. However, in the low concentration of Ang2 group, in spite of the existence of angiogenesis and new bone formation, the bone defects were not repaired. Furthermore, angiogenesis and osteogenesis were both obstructed in the control group. In conclusion, our study demonstrated that a high concentration of Ang2 promoted angiogenesis in tissue-engineered bone and improved repair of bone defects by inducing autophagy.

Plasma steroid profiling and response to trophins to illustrate intra-adrenal dynamics.

The importance of corticosteroids in cardiovascular and other chronic disease is recognised. In addition, plasma steroid precursor-to-product ratios are useful and convenient indirect indicators of efficiency of key steroidogenic enzymes (aldosterone synthase, 11ß-hydroxylase and 17α-hydroxylase). The use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) has enabled measurement of numerous corticosteroid compounds simultaneously. However, normal responses to trophins and variation in salt intake are not well described. This study examined these parameters in a large group of healthy volunteers. Sixty normotensive volunteers were recruited and underwent infusion of angiotensin II (AngII) and ACTH, following low- and high-salt diet. Measurement of plasma steroids at baseline and 30 min after infusion of trophin was carried out by LC-MS. As expected, plasma mineralocorticoid levels increased in response to salt restriction and were suppressed with salt loading; ACTH infusion increased all corticosteroids, while AngII increased mineralocorticoids and suppressed glucocorticoid production. ACTH increased S:F but decreased DOC:B, thus the S:F ratio is a more appropriate index of 11ß-hydroxylase efficiency. The B:F ratio increased following ACTH treatment and salt restriction. A larger proportion of plasma B than generally accepted may be derived from the zona glomerulosa and this ratio may be most informative of 17α-hydroxylase activity in salt-replete subjects. Although DOC:aldosterone, B:aldosterone and 18-hydroxyB:aldosterone should provide indices of aldosterone synthase efficiency, responses of individual compounds to trophins suggest that none of them accurately reflect this. Based on these data, aldosterone synthase activity is most accurately reflected by aldosterone concentration alone.

Angiopoietin-1 but not angiopoietin-2 induces IL-8 synthesis and release by human neutrophils.

We previously reported Tie2 receptor expression on human neutrophils, which promote chemotactic activities upon activation by both angiopoietins (Ang1 and Ang2). Moreover, we observed that neutrophil pretreatment with Ang1 or Ang2 enhances interleukin-8 (IL-8) chemotactic effect. Therefore, we assessed the capacity of Ang1 and/or Ang2 to modulate neutrophil IL-8 synthesis and release. Neutrophils isolated from healthy donors were stimulated in a time- (1-6 h) and concentration-(10(-10) -10(-8) M) dependent manner with both angiopoietins. IL-8 mRNA production was measured by RT-qPCR, whereas its protein synthesis and release from neutrophils was assessed by ELISA. Ang1 (10(-8) M) induced a significant and maximal increase of IL-8 mRNA (4.7-fold) within 1 h, and promoted maximal IL-8 protein synthesis (3.6-fold) and release (5.5-fold) within 2 h as compared to control PBS-treated neutrophils. Treatment with Ang2 alone did not modulate IL-8 synthesis or release, and its combination to Ang1 did not affect Ang1 activity. Neutrophil pretreatment with a protein synthesis inhibitor (CHX) increased IL-8 mRNA synthesis by 18-fold, and reduced Ang1-mediated IL-8 protein synthesis and release by 96% and 92%, respectively. Pretreatment with a transcription inhibitor (ActD) reduced IL-8 mRNA synthesis by 54% and IL-8 protein synthesis and release by 52% and 79%, respectively. Using specific kinase inhibitors, we observed that Ang1-driven IL-8 mRNA and protein synthesis is p42/44 MAPK-dependent and -independent from p38 MAPK and PI3K activity. Our study is the first to report the capacity of Ang1 (as opposed to Ang2) to promote neutrophil IL-8 synthesis and release through the activation of p42/44 MAPK pathway.

Experimental assessment of pro-lymphangiogenic growth factors in the treatment of post-surgical lymphedema following lymphadenectomy.

INTRODUCTION: Lymphedema is a frequent consequence of lymph node excision during breast cancer surgery. Current treatment options are limited mainly to external compression therapies to limit edema development. We investigated previously, post-surgical lymphedema in a sheep model following the removal of a single lymph node and determined that autologous lymph node transplantation has the potential to reduce or prevent edema development. In this report, we examine the potential of lymphangiogenic therapy to restore lymphatic function and reduce post-surgical lymphedema. METHODS: Lymphangiogenic growth factors (vascular endothelial growth factor-C (VEGF-C) and angiopoitein-2 (ANG-2)) were loaded into a gel-based drug delivery system (HAMC; a blend of hyaluronan and methylcellulose). Drug release rates and lymphangiogenic signaling in target endothelial cells were assessed in vitro and vascular permeability biocompatibility tests were examined in vivo. Following, the removal of a single popliteal lymph node, HAMC with the growth factors was injected into the excision site. Six weeks later, lymphatic functionality was assessed by injecting (125)Iodoine radiolabelled bovine serum albumin ((125)I-BSA) into prenodal vessels and measuring its recovery in plasma. Circumferential leg measurements were plotted over time and areas under the curves used to quantify edema formation. RESULTS: The growth factors were released over a two-week period in vitro by diffusion from HAMC, with 50% being released in the first 24 hours. The system induced lymphangiogenic signaling in target endothelial cells, while inducing only a minimal inflammatory response in sheep. Removal of the node significantly reduced lymphatic functionality (Nodectomy 1.9 ± 0.9, HAMC alone 1.7 ± 0.8) compared with intact groups (3.2 ± 0.7). There was no significant difference between the growth factor treatment group (2.3 ± 0.73) and the intact group. An increase in the number of regenerated lymphatic vessels at treatment sites was observed with fluoroscopy. Groups receiving HAMC plus growth factors displayed significantly reduced edema (107.4 ± 51.3) compared with non-treated groups (nodectomy 219.8 ± 118.7, and HAMC alone 162.6 ± 141). CONCLUSIONS: Growth factor therapy has the potential to increase lymphatic function and reduce edema magnitude in an animal model of lymphedema. The application of this concept to lymphedema patients warrants further examination.

Excess circulating angiopoietin-2 may contribute to pulmonary vascular leak in sepsis in humans.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a devastating complication of numerous underlying conditions, most notably sepsis. Although pathologic vascular leak has been implicated in the pathogenesis of ARDS and sepsis-associated lung injury, the mechanisms promoting leak are incompletely understood. Angiopoietin-2 (Ang-2), a known antagonist of the endothelial Tie-2 receptor, was originally described as a naturally occurring disruptor of normal embryonic vascular development otherwise mediated by the Tie-2 agonist angiopoietin-1 (Ang-1). We hypothesized that Ang-2 contributes to endothelial barrier disruption in sepsis-associated lung injury, a condition involving the mature vasculature. METHODS AND FINDINGS: We describe complementary human, murine, and in vitro investigations that implicate Ang-2 as a mediator of this process. We show that circulating Ang-2 is significantly elevated in humans with sepsis who have impaired oxygenation. We then show that serum from these patients disrupts endothelial architecture. This effect of sepsis serum from humans correlates with measured Ang-2, abates with clinical improvement, and is reversed by Ang-1. Next, we found that endothelial barrier disruption can be provoked by Ang-2 alone. This signal is transduced through myosin light chain phosphorylation. Last, we show that excess systemic Ang-2 provokes pulmonary leak and congestion in otherwise healthy adult mice. CONCLUSIONS: Our results identify a critical role for Ang-2 in disrupting normal pulmonary endothelial function.

Local delivery of angiopoietin-2 into the preovulatory follicle terminates the menstrual cycle in rhesus monkeys.

The angiopoietin (ANGPT)-receptor (TEK) system plays a crucial role in blood vessel formation and stability. Because the endogenous agonist ANGPT1, antagonist ANGPT2, and TEK are expressed in the primate ovary, experiments were designed to investigate their role at a critical time during tissue remodeling/ angiogenesis in the menstrual cycle (i.e., at midcycle during maturation, ovulation, and luteinization of the dominant follicle). Either vehicle, 20 microg of ANGPT1, 2 microg of ANGPT2 (low-dose), or 20 microg of ANGPT2 (high-dose) was injected directly into the preovulatory follicle of monkeys around the day (-1 to 0) of the midcycle estradiol (E2)/LH peak. Ovaries were evaluated on Day 3 postinjection for follicle rupture, and serum samples were analyzed for levels of E2 and progesterone. Similar to controls, ANGPT1 treatment was followed by ovulation, and elevated progesterone levels during the luteal phase. In contrast, high-dose ANGPT2 treatment prevented follicle rupture, and progesterone levels never rose above baseline in the subsequent 12 days. However, an E2 peak typically occurred 12 days postinjection. Laparoscopy detected a preovulatory follicle on the contralateral (noninjected) ovary. Progesterone levels subsequently increased above baseline in these animals. Thus, exogenous ANGPT2 disrupted maturation of the preovulatory follicle, preventing its ovulation and conversion into the corpus luteum. ANGPT antagonism eliminated the dominant structure, thereby resetting the ovarian cycle, with selection and maturation of the next preovulatory follicle occurring in a timely manner. The data are consistent with a critical role of the ANGPT-TIE1/TEK system in the ovary, notably at the late stages of follicle maturation during the menstrual cycle.