Louise L. Dunn

The Emerging Role of the Thioredoxin System in Angiogenesis

Although there have been a multitude of studies, the mechanisms of angiogenesis remain incompletely understood. Increasing evidence suggests that cellular redox homeostasis is an important regulator of angiogenesis. The thioredoxin (TRX) system functions as an endogenous antioxidant that can exert influence over endothelial cell function via modulation of cellular redox status. It has become apparent that the cytosolic TRX1 isoform participates in both canonical and novel angiogenic signaling pathways and may represent an avenue for therapeutic exploitation. Recent studies have further identified a role for the mitochondrial isoform TRX2 in ischemia-induced angiogenesis. TRX-interacting protein (TXNIP) is the endogenous inhibitor of TRX redox activity that has been implicated in growth factor-mediated angiogenesis. As TXNIP is strongly induced by glucose, this molecule could be of consequence to disordered angiogenesis manifest in diabetes mellitus. This review will focus on data implicating the TRX system in endothelial cell homeostasis and angiogenesis.

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

The melanoma tumor antigen, melanotransferrin (p97): a 25-year hallmark – from iron metabolism to tumorigenesis

Melanotransferrin (MTf) or melanoma tumor antigen p97 is a transferrin (Tf) homolog that is found predominantly bound to the cell membrane via a glycosyl phosphatidylinositol anchor. The molecule is a member of the Tf superfamily and binds iron through a single high-affinity iron(III)-binding site. Since its discovery on the plasma membrane of melanoma cells, the function of MTf has remained intriguing, particularly in relation to its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways, e.g., DNA synthesis, it was important to understand the function of MTf in the transport of this vital nutrient. MTf has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis and cell migration. However, recent studies using a knockout mouse and post-transcriptional gene silencing have demonstrated that MTf is not involved in iron metabolism, but plays a vital role in melanoma cell proliferation and tumorigenesis. In this review, we discuss the possible biological functions of MTf, particularly in relation to cancer.

New Insights into Intracellular Locations and Functions of Heme Oxygenase-1

SIGNIFICANCE Heme oxygenase-1 (HMOX1) plays a critical role in the protection of cells, and the inducible enzyme is implicated in a spectrum of human diseases. The increasing prevalence of cardiovascular and metabolic morbidities, for which current treatment approaches are not optimal, emphasizes the necessity to better understand key players such as HMOX1 that may be therapeutic targets. RECENT ADVANCES HMOX1 is a dynamic protein that can undergo post-translational and structural modifications which modulate HMOX1 function. Moreover, trafficking from the endoplasmic reticulum to other cellular compartments, including the nucleus, highlights that HMOX1 may play roles other than the catabolism of heme. CRITICAL ISSUES The ability of HMOX1 to be induced by a variety of stressors, in an equally wide variety of tissues and cell types, represents an obstacle for the therapeutic exploitation of the enzyme. Any capacity to modulate HMOX1 in cardiovascular and metabolic diseases should be tempered with an appreciation that HMOX1 may have an impact on cancer. Moreover, the potential for heme catabolism end products, such as carbon monoxide, to amplify the HMOX1 stress response should be considered. FUTURE DIRECTIONS A more complete understanding of HMOX1 modifications and the properties that they impart is necessary. Delineating these parameters will provide a clearer picture of the opportunities to modulate HMOX1 in human disease.

A Critical Role for Thioredoxin-Interacting Protein in Diabetes-Related Impairment of Angiogenesis

Impaired angiogenesis in ischemic tissue is a hallmark of diabetes. Thioredoxin-interacting protein (TXNIP) is an exquisitely glucose-sensitive gene that is overexpressed in diabetes. As TXNIP modulates the activity of the key angiogenic cytokine vascular endothelial growth factor (VEGF), we hypothesized that hyperglycemia-induced dysregulation of TXNIP may play a role in the pathogenesis of impaired angiogenesis in diabetes. In the current study, we report that high glucose–mediated overexpression of TXNIP induces a widespread impairment in endothelial cell (EC) function and survival by reducing VEGF production and sensitivity to VEGF action, findings that are rescued by silencing TXNIP with small interfering RNA. High glucose–induced EC dysfunction was recapitulated in normal glucose conditions by overexpressing either TXNIP or a TXNIP C247S mutant unable to bind thioredoxin, suggesting that TXNIP effects are largely independent of thioredoxin activity. In streptozotocin-induced diabetic mice, TXNIP knockdown to nondiabetic levels rescued diabetes-related impairment of angiogenesis, arteriogenesis, blood flow, and functional recovery in an ischemic hindlimb. These findings were associated with in vivo restoration of VEGF production to nondiabetic levels. These data implicate a critical role for TXNIP in diabetes-related impairment of ischemia-mediated angiogenesis and identify TXNIP as a potential therapeutic target for the vascular complications of diabetes.

High-density lipoproteins suppress chemokine expression and proliferation in human vascular smooth muscle cells

The inflammatory chemokines CCL2, CCL5, and CX3CL1 stimulate vascular smooth muscle cell (SMC) proliferation. High‐density lipoproteins (HDLs) exhibit potent cardioprotective and anti‐inflammatory properties. We therefore sought to determine the effect of reconstituted HDLs (rHDLs) on SMC chemokine expression and proliferation and elucidate the mechanisms. Preincubation of primary human SMCs with rHDLs containing apolipoprotein (apo)A‐I and phosphatidylcholine (20 μM, final apoA‐I concentration), before stimulation with TNF‐α, inhibited CCL2 (54%), CCL5 (38%), and CX3CL1 (33%) protein levels. The chemokine receptors CCR2 (29%) and CX3CR1 (22%) were also reduced by rHDLs. Incubation with rHDLs reduced the NF‐κB subunit p65 in the nucleus (39%) and phosphorylated IκBα (28%), both regulators of chemokine expression. Furthermore, rHDLs inhibited the upstream signaling proteins phosphoinositide 3‐kinase (37%) and phosphorylated Akt (pAkt, 49%). Incubation with rHDLs strikingly suppressed SMC proliferation (84%) and ERK phosphorylation (pERK, 29%). Finally, siRNA knockdown of the scavenger receptor SR‐B1 attenuated rHDL‐induced inhibition of SMC chemokine expression, p65, and proliferation, indicating that SR‐B1 plays a key role in mediating these effects. Thus, rHDLs reduce SMC chemokine expression (via NF‐κB/pAkt inhibition) and proliferation (via pERK inhibition). This has important implications for preventing the pathogenesis of neointimal hyperplasia, the main cause of early vein graft/stent failure.—Van der Vorst, E. P. C., Vanags, L. Z., Dunn, L. L., Prosser, H. C., Rye, K.‐A., Bursill, C. A. High‐density lipoproteins suppress chemokine expression and proliferation in human vascular smooth muscle cells. FASEB J. 27, 1413–1425 (2013). www.fasebj.org

Multifunctional Regulation of Angiogenesis by High Density Lipoproteins

AIMS High-density lipoproteins (HDL) exert striking anti-inflammatory effects and emerging evidence suggests that they may augment ischaemia-mediated neovascularization. We sought to determine whether HDL conditionally regulates angiogenesis, depending on the pathophysiological context by (i) inhibiting inflammation-induced angiogenesis, but also; (ii) enhancing ischaemia-mediated angiogenesis. METHODS AND RESULTS Intravenously delivered apolipoprotein (apo) A-I attenuated neovascularization in the murine femoral collar model of inflammation-induced angiogenesis, compared with phosphate-buffered saline infused C57BL6/J mice (58%), P < 0.05. Conversely, apoA-I delivery augmented neovessel formation (75%) and enhanced blood perfusion (45%) in the murine hindlimb ischaemia model, P < 0.05. Reconstituted HDL (rHDL) was tested on key angiogenic cell functions in vitro. rHDL inhibited human coronary artery endothelial cell migration (37.9 and 76.9%), proliferation (15.7 and 40.4%), and tubulogenesis on matrigel (52 and 98.7%) when exposed to two inflammatory stimuli: tumour necrosis factor-α (TNF-α) and macrophage-conditioned media (MCM). In contrast, rHDL significantly augmented hypoxia-stimulated migration (36.9%), proliferation (135%), and tubulogenesis (22.9%), P < 0.05. Western blot and RT-PCR analyses revealed that these divergent actions of rHDL were associated with conditional regulation of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and VEGF receptor 2, which were attenuated in response to TNF-α (40.4, 41.0, and 33.2%) and MCM (72.5, 30.7, and 69.5%), but augmented by rHDL in hypoxia (39.8, 152.6, and 15.7%%), all P < 0.05. CONCLUSION HDL differentially regulates angiogenesis dependent upon the pathophysiological setting, characterized by suppression of inflammation-associated angiogenesis, and conversely, by the enhancement of hypoxia-mediated angiogenesis. This has significant implications for therapeutic modulation of neovascularization.

High-Density Lipoproteins Rescue Diabetes-Impaired Angiogenesis via Scavenger Receptor Class B Type I

Disordered neovascularization and impaired wound healing are important contributors to diabetic vascular complications. We recently showed that high-density lipoproteins (HDLs) enhance ischemia-mediated neovascularization, and mounting evidence suggests HDL have antidiabetic properties. We therefore hypothesized that HDL rescue diabetes-impaired neovascularization. Streptozotocin-induced diabetic mice had reduced blood flow recovery and neovessel formation in a hindlimb ischemia model compared with nondiabetic mice. Reconstituted HDL (rHDL) infusions in diabetic mice restored blood flow recovery and capillary density to nondiabetic levels. Topical rHDL application rescued diabetes-impaired wound closure, wound angiogenesis, and capillary density. In vitro, rHDL increased key mediators involved in hypoxia-inducible factor-1α (HIF-1α) stabilization, including the phosphoinositide 3-kinase/Akt pathway, Siah1, and Siah2, and suppressed the prolyl hydroxylases (PHD) 2 and PHD3. rHDL rescued high glucose–induced impairment of tubulogenesis and vascular endothelial growth factor (VEGF) A protein production, a finding associated with enhanced phosphorylation of proangiogenic mediators VEGF receptor 2 and endothelial nitric oxide synthase. Siah1/2 small interfering RNA knockdown confirmed the importance of HIF-1α stability in mediating rHDL action. Lentiviral short hairpin RNA knockdown of scavenger receptor class B type I (SR-BI) in vitro and SR-BI−/− diabetic mice in vivo attenuated rHDL rescue of diabetes-impaired angiogenesis, indicating a key role for SR-BI. These findings provide a greater understanding of the vascular biological effects of HDL, with potential therapeutic implications for diabetic vascular complications.

Stability of a Therapeutic Layer of Immobilized Recombinant Human Tropoelastin on a Plasma-Activated Coated Surface

ABSTRACTPurposeTo modify blood-contacting stainless surfaces by covalently coating them with a serum-protease resistant form of tropoelastin (TE). To demonstrate that the modified TE retains an exposed, cell-adhesive C-terminus that persists in the presence of blood plasma proteases.MethodsRecombinant human TE and a point mutant variant (R515A) of TE were labeled with 125Iodine and immobilized on plasma-activated stainless steel (PAC) surfaces. Covalent attachment was confirmed using rigorous detergent washing. As kallikrein and thrombin dominate the serum degradation of tropoelastin, supraphysiological levels of these proteases were incubated with covalently bound TE and R515A, then assayed for protein levels by radioactivity detection. Persistence of the C-terminus was assessed by ELISA.ResultsTE was significantly retained covalently on PAC surfaces at 88 ± 5% and 71 ± 5% after treatment with kallikrein and thrombin, respectively. Retention of R515A was 100 ± 1.3% and 87 ± 2.3% after treatment with kallikrein and thrombin, respectively, representing significant improvements over TE. The functionally important C-terminus was cleaved in wild-type TE but retained by R515A.ConclusionsProtein persists in the presence of human kallikrein and thrombin when covalently immobilized on metal substrata. R515A displays enhanced protease resistance and retains the C-terminus presenting a protein interface that is viable for blood-contacting applications.

The relationship between endothelial progenitor cell populations and epicardial and microvascular coronary disease-a cellular, angiographic and physiologic study.

Background Endothelial progenitor cells (EPCs) are implicated in protection against vascular disease. However, studies using angiography alone have reported conflicting results when relating EPCs to epicardial coronary artery disease (CAD) severity. Moreover, the relationship between different EPC types and the coronary microcirculation is unknown. We therefore investigated the relationship between EPC populations and coronary epicardial and microvascular disease. Methods Thirty-three patients with a spectrum of isolated left anterior descending artery disease were studied. The coronary epicardial and microcirculation were physiologically interrogated by measurement of fractional flow reserve (FFR), index of microvascular resistance (IMR) and coronary flow reserve (CFR). Two distinct EPC populations (early EPC and late outgrowth endothelial cells [OECs]) were isolated from these patients and studied ex vivo. Results There was a significant inverse relationship between circulating OEC levels and epicardial CAD severity, as assessed by FFR and angiography (r = 0.371, p = 0.04; r = -0.358, p = 0.04; respectively). More severe epicardial CAD was associated with impaired OEC migration and tubulogenesis (r = 0.59, p = 0.005; r = 0.589, p = 0.004; respectively). Patients with significant epicardial CAD (FFR<0.75) had lower OEC levels and function compared to those without hemodynamically significant stenoses (p<0.05). In contrast, no such relationship was seen for early EPC number and function, nor was there a relationship between IMR and EPCs. There was a significant relationship between CFR and OEC function. Conclusions EPC populations differ in regards to their associations with CAD severity. The number and function of OECs, but not early EPCs, correlated significantly with epicardial CAD severity. There was no relationship between EPCs and severity of coronary microvascular disease.