Can We Advance Macroscopic Quantum Systems Outside the Framework of Complex Decoherence Theory?

Macroscopic quantum systems (MQS) are macroscopic systems driven by quantum rather than classical mechanics, a long studied area with minimal success till recently. Harnessing the benefits of quantum mechanics on a macroscopic level would revolutionize fields ranging from telecommunication to biology, the latter focused on here for reasons discussed. Contrary to misconceptions, there are no known physical laws that prevent the development of MQS. Instead, they are generally believed universally lost in complex systems from environmental entanglements (decoherence). But we argue success is achievable MQS with decoherence compensation developed, naturally or artificially, from top-down rather current reductionist approaches. This paper advances the MQS field by a complex systems approach to decoherence. First, why complex system decoherence approaches (top-down) are needed is discussed. Specifically, complex adaptive systems (CAS) are not amenable to reductionist models (and their master equations) because of emergent behaviour, approximation failures, not accounting for quantum compensatory mechanisms, ignoring path integrals, and the subentity problem. In addition, since MQS must exist within the context of the classical world, where rapid decoherence and prolonged coherence are both needed. Nature has already demonstrated this for quantum subsystems such as photosynthesis and magnetoreception. Second, we perform a preliminary study that illustrates a top-down approach to potential MQS. In summary, reductionist arguments against MQS are not justifiable. It is more likely they are not easily detectable in large intact classical systems or have been destroyed by reductionist experimental set-ups. This complex systems decoherence approach, using top down investigations, is critical to paradigm shifts in MQS research both in biological and non-biological systems.

QHREDGS enhances tube formation, metabolism and survival of endothelial cells in collagen-chitosan hydrogels.

Cell survival in complex, vascularized tissues, has been implicated as a major bottleneck in advancement of therapies based on cardiac tissue engineering. This limitation motivates the search for small, inexpensive molecules that would simultaneously be cardio-protective and vasculogenic. Here, we present peptide sequence QHREDGS, based upon the fibrinogen-like domain of angiopoietin-1, as a prime candidate molecule. We demonstrated previously that QHREDGS improved cardiomyocyte metabolism and mitigated serum starved apoptosis. In this paper we further demonstrate the potency of QHREDGS in its ability to enhance endothelial cell survival, metabolism and tube formation. When endothelial cells were exposed to the soluble form of QHREDGS, improvements in endothelial cell barrier functionality, nitric oxide production and cell metabolism (ATP levels) in serum starved conditions were found. The functionality of the peptide was then examined when conjugated to collagen-chitosan hydrogel, a potential carrier for in vivo application. The presence of the peptide in the hydrogel mitigated paclitaxel induced apoptosis of endothelial cells in a dose dependent manner. Furthermore, the peptide modified hydrogels stimulated tube-like structure formation of encapsulated endothelial cells. When integrin αvß3 or α5ß1 were antibody blocked during cell encapsulation in peptide modified hydrogels, tube formation was abolished. Therefore, the dual protective nature of the novel peptide QHREDGS may position this peptide as an appealing augmentation for collagen-chitosan hydrogels that could be used for biomaterial delivered cell therapies in the settings of myocardial infarction.

Endostatin lowers blood pressure via nitric oxide and prevents hypertension associated with VEGF inhibition.

Antiangiogenesis therapy has become a vital part of the armamentarium against cancer. Hypertension is a dose-limiting toxicity for VEGF inhibitors. Thus, there is a pressing need to address the associated adverse events so these agents can be better used. The hypertension may be mediated by reduced NO bioavailability resulting from VEGF inhibition. We proposed that the hypertension may be prevented by coadministration with endostatin (ES), an endogenous angiogenesis inhibitor with antitumor effects shown to increase endothelial NO production in vitro. We determined that Fc-conjugated ES promoted NO production in endothelial and smooth muscle cells. ES also lowered blood pressure in normotensive mice and prevented hypertension induced by anti-VEGF antibodies. This effect was associated with higher circulating nitrate levels and was absent in eNOS-knockout mice, implicating a NO-mediated mechanism. Retrospective study of patients treated with ES in a clinical trial revealed a small but significant reduction in blood pressure, suggesting that the findings may translate to the clinic. Coadministration of ES with VEGF inhibitors may offer a unique strategy to prevent drug-related hypertension and enhance antiangiogenic tumor suppression.

Contrary effects of the receptor tyrosine kinase inhibitor vandetanib on constitutive and flow-stimulated nitric oxide elaboration in humans.

Vascular endothelial growth factor regulates neoplastic angiogenesis through production of endothelium-derived NO. We performed a prospective evaluation of vascular function during treatment with vandetanib, a vascular endothelial growth receptor 2 and 3 receptor tyrosine kinase inhibitor, to determine the effects of vascular endothelial growth receptor signal interruption on endothelial function in humans. Seventeen patients with stage IV breast cancer received dose-escalated vandetanib in combination with low-dose oral chemotherapy. We measured blood pressure, systemic nitrate/nitrite levels, and brachial artery vascular function. In vitro analyses of cultured endothelial cells were performed to determine the effect of vandetanib on NO production, akt(473) phosphorylation, and endothelial NO synthase protein content and membrane localization. Vandetanib treatment for 6 weeks significantly increased blood pressure, decreased resting brachial artery diameter, and decreased plasma systemic nitrate/nitrite levels compared with baseline. Flow-mediated vasodilation was preserved, and no change was noted in nitroglycerin-mediated vasodilation. In vitro, endothelial cell nitrite levels and akt(473) phosphorylation were reduced and vascular endothelial growth receptor 2 levels did not change, but endothelial NO synthase membrane concentration doubled. Vandetanib reduces constitutive NO production and increases blood pressure, yet flow-stimulated NO bioavailability was preserved. Changes in vascular function with tyrosine kinase inhibition are complex and require further study in humans.

Photocrosslinkable chitosan modified with angiopoietin-1 peptide, QHREDGS, promotes survival of neonatal rat heart cells.

Myocardial infarction (MI) results in the death of cardiomyocytes (CM), which causes scar formation and pathological remodeling of the heart. The delivery of healthy myocytes or bone marrow cells reduces pathological remodeling after MI, however, current cell injection methods have low cell survival rates and high cell loss. The main objective of this work was to develop a novel hydrogel that can promote survival of CMs. Photocrosslinkable azidobenzoic acid modified chitosan (Az-chitosan) was conjugated with the angiopoietin-1-derived peptide, QHREDGS. This novel peptide is thought to mediate attachment and survival responses of CM to angiopoietin-1 via integrin binding. Thin layers of Az-chitosan, Az-chitosan-QHREDGS, and Az-chitosan-DGQESHR (scrambled peptide control) were spin coated on glass slides and photocrosslinked with application of UV light (365 nm). Neonatal rat heart cells cultured up to 5 days, demonstrated significantly higher attachment and viability on Az-chitosan-QHREDGS compared to cells on other hydrogel controls. Surfaces were also stained for the CM-specific marker troponin I, demonstrating significantly higher percentage of CMs on Az-chitosan-QHREDGS compared to Az-chitosan. The cells cultivated on Az-chitosan-QHREDGS demonstrated significantly lower levels of caspase 3/7 activation after taxol treatment in comparison to cells cultivated on the control hydrogels, glass substrate, or Az-chitosan linked to RGD, an established integrin binding peptide that did not protect against apoptosis. Thus, Az-chitosan-QHREDGS supports attachment and survival of neonatal rat heart cells.

Integrin binding angiopoietin-1 monomers reduce cardiac hypertrophy.

Angiopoietins were thought to be endothelial cell-specific via the tie2 receptor. We showed that angiopoietin-1 (ang1) also interacts with integrins on cardiac myocytes (CMs) to increase survival. Because ang1 monomers bind and activate integrins (not tie2), we determined their function in vivo. We examined monomer and multimer expressions during physiological and pathological cardiac remodeling and overexpressed ang1 monomers in phenylephrine-induced cardiac hypertrophy. Cardiac ang1 levels (mRNA, protein) increased during postnatal development and decreased with phenylephrine-induced cardiac hypertrophy, whereas tie2 phosphorylations were unchanged. We found that most or all of the changes during cardiac remodeling were in monomers, offering an explanation for unchanged tie2 activity. Heart tissue contains abundant ang1 monomers and few multimers (Western blotting). We generated plasmids that produce ang1 monomers (ang1-256), injected them into mice, and confirmed cardiac expression (immunohistochemistry, RT-PCR). Ang1 monomers localize to CMs, smooth muscle cells, and endothelial cells. In phenylephrine-induced cardiac hypertrophy, ang1-256 reduced left ventricle (LV)/tibia ratios, fetal gene expressions (atrial and brain natriuretic peptides, skeletal actin, beta-myosin heavy chain), and fibrosis (collagen III), and increased LV prosurvival signaling (akt, MAPK(p42/44)), and AMPK(T172). However, tie2 phosphorylations were unchanged. Ang1-256 increased integrin-linked kinase, a key regulator of integrin signaling and cardiac health. Collectively, these results suggest a role for ang1 monomers in cardiac remodeling.

Cardiotoxicity associated with tyrosine kinase inhibitor sunitinib.

BACKGROUND: Sunitinib, a multitargeted tyrosine-kinase inhibitor, which is approved by both US and European Commission regulatory agencies for clinical use, extends survival of patients with metastatic renal-cell carcinoma and gastrointestinal stromal tumours, but concerns have arisen about its cardiac safety. We therefore assessed the cardiovascular risk associated with sunitinib in patients with metastatic gastrointestinal stromal tumours. METHODS: We retrospectively reviewed all cardiovascular events in 75 patients with imatinib-resistant, metastatic, gastrointestinal stromal tumours who had been enrolled in a phase I/II trial investigating the efficacy of sunitinib. The composite cardiovascular endpoint was cardiac death, myocardial infarction, and congestive heart failure. We also examined sunitinib's effects on left ventricular ejection fraction (LVEF) and blood pressure. We investigated potential mechanisms of sunitinib-associated cardiac effects by studies in isolated rat cardiomyocytes and in mice. FINDINGS: Eight of 75 (11%) patients given repeating cycles of sunitinib in the phase I/II trial had a cardiovascular event, with congestive heart failure recorded in six of 75 (8%). Ten of 36 (28%) patients treated at the approved sunitinib dose had absolute LVEF reductions in ejection fraction (EF) of at least 10%, and seven of 36 (19%) had LVEF reductions of 15 EF% or more. Sunitinib induced increases in mean systolic and diastolic blood pressure, and 35 of 75 (47%) individuals developed hypertension (>150/100 mm Hg). Congestive heart failure and left ventricular dysfunction generally responded to sunitinib being withheld and institution of medical management. Sunitinib caused mitochondrial injury and cardiomyocyte apoptosis in mice and in cultured rat cardiomyocytes. INTERPRETATION: Left ventricular dysfunction might be due, in part, to direct cardiomyocyte toxicity, exacerbated by hypertension. Patients treated with sunitinib should be closely monitored for hypertension and LVEF reduction, especially those with a history of coronary artery disease or cardiac risk factors.

Vascular progenitor cells isolated from human embryonic stem cells give rise to endothelial and smooth muscle like cells and form vascular networks in vivo.

We report that human embryonic stem cells contain a population of vascular progenitor cells that have the ability to differentiate into endothelial-like and smooth muscle (SM)-like cells. Vascular progenitor cells were isolated from EBs grown in suspension for 10 days and were characterized by expression of the endothelial/hematopoietic marker CD34 (CD34+ cells). When these cells are subsequently cultured in EGM-2 (endothelial growth medium) supplemented with vascular endothelial growth factor-165 (50 ng/mL), they give rise to endothelial-like cells characterized by a cobblestone cell morphology, expression of endothelial markers (platelet endothelial cell-adhesion molecule-1, CD34, KDR/Flk-1, vascular endothelial cadherin, von Willebrand factor), incorporation of acetylated low-density lipoprotein, and formation of capillary-like structures when placed in Matrigel. In contrast, when CD34+ cells are cultured in EGM-2 supplemented with platelet-derived growth factor-BB (50 ng/mL), they give rise to SM-like cells characterized by spindle-shape morphology, expression of SM cell markers (alpha-SM actin, SM myosin heavy chain, calponin, caldesmon, SM alpha-22), and the ability to contract and relax in response to common pharmacological agents such as carbachol and atropine but rarely form capillary-like structures when placed in Matrigel. Implantation studies in nude mice show that both cell types contribute to the formation of human microvasculature. Some microvessels contained mouse blood cells, which indicates functional integration with host vasculature. Therefore, the vascular progenitors isolated from human embryonic stem cells using methods established in the present study could provide a means to examine the mechanisms of endothelial and SM cell development, and they could also provide a potential source of cells for vascular tissue engineering.

Endostatin inhibits the growth of endometriotic lesions but does not affect fertility.

OBJECTIVE: To determine whether endometriosis can be treated with the angiogenesis inhibitor endostatin and the effect of this treatment on fertility and reproduction. DESIGN: Pharmacologic intervention in a surgically induced model of endometriosis and in female mice undergoing mating. SETTING: Animal research facility. ANIMAL(S): Eight-week-old, female C57BL/6 and SCID mice. INTERVENTION(S): After implantation of autologous endometrium, mice received endostatin or the vehicle-matched control for 4 weeks. For the reproductive function study, mice receiving endostatin or vehicle were mated and reproductive functions were observed. MAIN OUTCOME MEASURE(S): Growth of endometriotic lesions after 4 weeks of treatment; estrous cycling, corpus luteum formation, serum hormone levels, and mating time as fertility measures; and pregnancy rates, length of pregnancy, fetal vitality, number, and outcome of litter as reproductive measures. RESULT(S): Endostatin suppressed the growth of endometriotic lesions by 47% compared with controls. Estrous cycling and corpus luteum formation were normal in both groups. Female mice receiving endostatin were as fertile as mice receiving vehicle, had normal pregnancies, and delivered the same number of pups. The offspring were healthy without teratogenic stigmata and reproduced normally themselves. CONCLUSION(S): Antiangiogenic therapy with endostatin may present a promising novel, nontoxic therapeutic option for patients with endometriosis.

Angiopoietin-1 promotes cardiac and skeletal myocyte survival through integrins.

Cardiac myocyte loss, regardless of insult, can trigger compensatory myocardial remodeling leading to heart failure. Identifying mediators of cardiac myocyte survival may advance clinical efforts toward myocardial preservation. Angiopoietin-1 limits ischemia-induced cardiac injury. This benefit is ascribed to angiogenesis because the receptor, tie2, is largely endothelial-specific. We propose that direct, non-tie2 interactions of angiopoietin-1 on cardiac myocytes contribute to this cardioprotection. We found that mouse C2C12 skeletal myocytes lack tie2, yet dose-dependently adhered to angiopoietin-1 and angiopoietin-2 similarly to laminin, fibronectin, vitronectin, and more than to collagen-I, -III, and -IV. Adhesion was divalent cation-mediated (Mn2+, Ca2+, not Mg2+), blocked with EDTA/EGTA, RGD-based peptides, and select integrin subunit antibodies. Similar findings were obtained with human skeletal myocytes (HSMs) and freshly isolated rat neonatal cardiac myocytes (NCMs). Furthermore, angiopoietin-1 conferred significant survival advantage exceeding that of most cell matrices, which was not fully explained by differences in cell adhesion. Angiopoietin-1 promoted survival of serum-starved C2C12, HSM, and NCM (MTT, trypan blue) and prevented taxol-induced apoptosis (caspase-3). Immobilized and soluble angiopoietin-1 phosphorylated Akt(S473) and MAPK(p42/44), (not FAK(Y397)) in C2C12 more than in endothelial cells and more than did angiopoietin-2 or cell matrices. EDTA, RGD-based peptides, and some integrin antibodies blocked these responses. Angiopoietin-1 activated HSM and NCM Akt(S473) and MAPK(p42/44) survival pathways. We propose that this novel function contributes to developmental and cardioprotective actions of angiopoietin-1 presently attributed to vascular effects alone. Angiopoietin-1 may prove therapeutically valuable in cardiac remodeling by supporting myocyte viability and preserving pump function. The full text of this article is available online at http://circres.ahajournals.org.

Nonsteroidal antiinflammatory drugs differentially suppress endometriosis in a murine model.

OBJECTIVE: To determine whether nonsteroidal antiinflammatory drugs (NSAIDs) affect the establishment and progression of endometriotic lesions in a murine model. DESIGN: Pharmacologic intervention in a surgically induced murine model of abdominal/peritoneal endometriosis. SETTING: Animal research facility. PATIENT(S): Eight-week-old, female C57BL/6 mice. INTERVENTION(S): After implantation of autologous endometrium, mice were randomized into groups and treated with one of several NSAIDs or the vehicle-matched control for 4 weeks. MAIN OUTCOME MEASURE(S): Establishment, growth, and total burden of endometriotic lesions. RESULT(S): The NSAIDs differentially inhibited lesion establishment and growth, resulting in significantly reduced disease burden. Compared with controls (5.7 +/- 2.3 mm(2)), lesion burden was reduced by celecoxib (1.3 +/- 1.2 mm(2)), indomethacin (1.4 +/- 1.4 mm(2)), naproxen (2.7 +/- 1.2 mm(2)), sulindac (3.1 +/- 1.5 mm(2)), rofecoxib (3.4 +/- 3.0 mm(2)), and ibuprofen (4.1 +/- 1.4 mm(2)). In contrast, aspirin (5.9 +/- 1.2 mm(2)) had no statistically significant effect. Uninterrupted estrus cycling was confirmed by vaginal exams and smears in celecoxib-treated mice. CONCLUSION(S): Chronic administration of certain NSAIDs limits the progression of endometriosis in this murine model. The data suggest that NSAID selection in the treatment of endometriosis should be extended beyond pain management to maximize the inhibitory effect on disease burden.

Adipose tissue growth and regression are regulated by angiopoietin-1.

Adipose tissue is unique in its plasticity, capacity for vascular remodeling, and susceptibility to angiogenesis inhibitors. We hypothesize that these characteristics are enabled by maintaining relatively immature adipose vessels to facilitate vascular/tissue remodeling. We examined the vascular maturation regulators, angiopoietin-1, angiopoietin-2, and tie2 receptor, under different weight-modifying conditions. Adipocytes expressed angiopoietin-1, while adipose endothelial cells expressed angiopoietin-2 and tie2. Adipose tissue growth/regression were associated with decreased angiopoietin-1 mRNA and protein, and tie2 phosphorylation. Angiopoietin-2 and tie2 mRNA levels were stable. Angiopoietin-1 mRNA levels inversely correlated with the rates of change in body weight, independent of the direction (weight gain, loss) or etiology (TNP-470, leptin, and diet restriction) of the weight shift. Obese mice injected with ang1/pcDNA had reduced rates of weight gain and fat pad weights, regardless of the route of plasmid administration (subcutaneous, intramuscular, and intravenous). Thus, angiopoietin-1 may regulate adipose tissue growth, suggesting that vascular maturation alters tissue plasticity.

Endothelial-directed hepatic regeneration after partial hepatectomy.

OBJECTIVE: To determine the role of the microvascular endothelium in the regulation of regenerating liver mass after partial hepatectomy. SUMMARY BACKGROUND DATA: Angiogenesis is critical for both pathologic and physiologic processes. The ability of certain tissues, such as the liver, kidney, and spleen, to regenerate after injury is poorly understood. The liver will regenerate to its normal mass within 8 days of surgical excision. Because the authors have previously shown that the endothelial cell regulates tumor mass, we hypothesized that normal adult organ mass is also controlled by the endothelial cell. METHODS: Two-thirds partial hepatectomy was performed in 7- to 8-week-old C57 BL/6 mice, followed by systemic treatment with either the angiogenesis stimulator basic fibroblast growth factor (bFGF) (1 microg/g/d intraperitoneal) or the angiogenesis inhibitor TNP-470 (30 mg/kg/qod subcutaneous). Groups of three mice were then euthanized on postoperative days 2, 4, 6, and 8, and the livers were weighed and analyzed by immunohistochemistry. RESULTS: bFGF accelerated hepatic regeneration by 42%, 19%, 16%, and 16% on postoperative days 2, 4, 6, and 8, respectively. TNP-470 inhibited hepatic regeneration by 46%, 74%, 67%, and 64% on postoperative days 2, 4, 6, and 8, respectively. Immunohistochemistry revealed that bFGF and TNP-470 primarily affected the endothelial compartment. Specifically, bFGF increased endothelial proliferation and decreased endothelial apoptosis. TNP-470, in contrast, inhibited endothelial cell proliferation. The cessation of the regenerative process correlated with a decrease in endothelial proliferation and an increase in endothelial apoptosis. CONCLUSIONS: The systemic administration of angiogenesis agents modulates the regeneration of hepatic mass primarily by affecting endothelial cell proliferation or apoptosis. Endothelial cell apoptosis is associated with the cessation of the regenerative process in control mice. These results suggest that the endothelial cell is one of the key mediators of regenerating adult tissue mass in this partial hepatectomy model.

Adipose tissue mass can be regulated through the vasculature.

Tumor growth is angiogenesis dependent. We hypothesized that nonneoplastic tissue growth also depends on neovascularization. We chose adipose tissue as an experimental system because of its remodeling capacity. Mice from different obesity models received anti-angiogenic agents. Treatment resulted in dose-dependent, reversible weight reduction and adipose tissue loss. Marked vascular remodeling was evident in adipose tissue sections, which revealed decreased endothelial proliferation and increased apoptosis in treated mice compared with controls. Continuous treatment maintained mice near normal body weights for age without adverse effects. Metabolic adaptations in food intake, metabolic rate, and energy substrate utilization were associated with anti-angiogenic weight loss. We conclude that adipose tissue mass is sensitive to angiogenesis inhibitors and can be regulated by its vasculature.

Effects of oxygen on engineered cardiac muscle.

Concentration gradients associated with the in vitro cultivation of engineered tissues that are vascularized in vivo result in the formation of only a thin peripheral tissue-like region (e.g., approximately 100 microm for engineered cardiac muscle) around a relatively cell-free interior. We previously demonstrated that diffusional gradients within engineered cardiac constructs can be minimized by direct perfusion of culture medium through the construct. In the present study, we measured the effects of medium perfusion rate and local oxygen concentration (p(O2)) on the in vitro reconstruction of engineered cardiac muscle. Neonatal rat cardiomyocytes were seeded onto biodegradable polymer scaffolds (fibrous discs, 1.1 cm diameter x 2 mm thick, made of polyglycolic acid, 24 x 10(6) cells per scaffold). The resulting cell-polymer constructs were cultured for a total of 12 days in serially connected cartridges (n = 1-8), each containing one construct directly perfused with culture medium at a flow rate of 0.2-3.0 mL/min. In all groups, oxygen concentration decreased due to cell respiration, and depended on construct position in the series and medium flow rate. Higher perfusion rates and higher p(O2) correlated with more aerobic cell metabolism, and higher DNA and protein contents. Constructs cultured at p(O2) of 160 mm Hg had 50% higher DNA and protein contents, markedly higher expression of sarcomeric alpha-actin, better organized sarcomeres and cell junctions, and 4.5-fold higher rate of cell respiration as compared to constructs cultured at p(O2) of 60 mm Hg. Contraction rates of the corresponding cardiac cell monolayers were 40% higher at p(O2) of 160 than 60 mm Hg. The control of oxygen concentration in cell microenvironment can thus improve the structure and function of engineered cardiac muscle. Experiments of this kind can form a basis for controlled studies of the effects of oxygen on the in vitro development of engineered tissues.

Perfusion improves tissue architecture of engineered cardiac muscle.

Cardiac muscle with a certain threshold thickness, uniformity of tissue architecture, and functionality would expand the therapeutic options currently available to patients with congenital or acquired cardiac defects. Cardiac constructs cultured in well-mixed medium had an approximately 100-microm-thick peripheral tissue-like region around a relatively cell-free interior, a structure consistent with the presence of concentration gradients within the tissue. We hypothesized that direct perfusion of cultured constructs can reduce diffusional distances for mass transport, improve control of oxygen, pH, nutrients and metabolites in the cell microenvironment, and thereby increase the thickness and spatial uniformity of engineered cardiac muscle. To test this hypothesis, constructs (9.5-mm-diameter, 2-mm-thick discs) based on neonatal rat cardiac myocytes and fibrous polyglycolic acid scaffolds were cultured either directly perfused with medium or in control spinner flasks. Perfusion improved the spatial uniformity of cell distribution and enhanced the expression of cardiac-specific markers, presumably due to the improved control of local microenvironmental conditions within the forming tissue. Medium perfusion could thus be utilized to better mimic the transport conditions within native cardiac muscle and enable in vitro engineering of cardiac constructs with clinically useful thicknesses.