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1.  Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis 
Nature materials  2016;15(6):669-678.
We report the fabrication of a scaffold (hereafter referred to as AngioChip) that supports the assembly of parenchymal cells on a mechanically tunable matrix surrounding a perfusable, branched, three-dimensional microchannel network coated with endothelial cells. The design of AngioChip decouples the material choices for the engineered vessel network and for cell seeding in the parenchyma, enabling extensive remodelling while maintaining an open-vessel lumen. The incorporation of nanopores and micro-holes in the vessel walls enhances permeability, and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation. We also show that vascularized hepatic tissues and cardiac tissues engineered by using AngioChips process clinically relevant drugs delivered through the vasculature, and that millimeter-thick cardiac tissues can be engineered in a scalable manner. Moreover, we demonstrate that AngioChip cardiac tissues implanted via direct surgical anastomosis to the femoral vessels of rat hindlimbs establish immediate blood perfusion.
PMCID: PMC4879054  PMID: 26950595
2.  Prevalence and clinical features of adverse food reactions in Portuguese adults 
Only one previous study, via telephone call, on the prevalence of self-reported food allergies has been performed in Portugal, in a small sample of adults. The objective of this study was to determine the prevalence of self-reported and probable food allergy, analyze the clinical features and involved foods in Portuguese adults.
Population-based, cross-sectional study performed in various healthcare centres from central Portugal. All 1436 randomly selected individuals (median age: 45 years, 50.6 % female) replied to a validated food allergy questionnaire by phone. Those who reported an adverse food reaction were invited to come to the hospital, where clinical history was taken, skin prick (SPT) and prick-prick skin (SPPT) tests were performed and food allergen-specific IgE levels (sIgE) were determined. An open oral challenge was performed in selected cases. Cases of positive clinical history of immediate (up to 2 h after ingestion) reaction in association with positive food sIgE levels and/or skin prick tests were classified as IgE-associated probable food allergy. Cases of positive clinical history of delayed (more than 2 h after ingestion) and negative food sIgE levels independently of positive SPT or SPPT results were classified as non-IgE associated probable food allergy.
The prevalence of probable food allergy in our sample was 1 %, with shellfish and fish as the most frequently implicated foods. IgE-mediated probable food allergy occurred in 0.71 % of cases, with shellfish, peanut and nuts mainly involved. Cutaneous symptoms were most frequently reported. Prevalence values and food types were discrepant between self-reported and probable food allergies.
The prevalence of probable food allergies in Portuguese adults is low, is mostly related to shellfish, peanut and nuts and most frequently involves cutaneous symptoms.
Electronic supplementary material
The online version of this article (doi:10.1186/s13223-016-0139-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4975906  PMID: 27499765
Adverse food reaction; Food allergy; Adults; Prevalence; Cutaneous tests; Open food challenge
3.  Safety profile of solid lipid nanoparticles loaded with rosmarinic acid for oral use: in vitro and animal approaches 
Rosmarinic acid (RA) possesses several protective bioactivities that have attracted increasing interest by nutraceutical/pharmaceutical industries. Considering the reduced bioavailability after oral use, effective (and safe) delivery systems are crucial to protect RA from gastrointestinal degradation. This study aims to characterize the safety profile of solid lipid nanoparticles produced with Witepsol and Carnauba waxes and loaded with RA, using in vitro and in vivo approaches, focused on genotoxicity and cytotoxicity assays, redox status markers, hematological and biochemical profile, liver and kidney function, gut bacterial microbiota, and fecal fatty acids composition. Free RA and sage extract, empty nanoparticles, or nanoparticles loaded with RA or sage extract (0.15 and 1.5 mg/mL) were evaluated for cell (lymphocytes) viability, necrosis and apoptosis, and antioxidant/prooxidant effects upon DNA. Wistar rats were orally treated for 14 days with vehicle (control) and with Witepsol or Carnauba nanoparticles loaded with RA at 1 and 10 mg/kg body weight/d. Blood, urine, feces, and several tissues were collected for analysis. Free and loaded RA, at 0.15 mg/mL, presented a safe profile, while genotoxic potential was found for the higher dose (1.5 mg/mL), mainly by necrosis. Our data suggest that both types of nanoparticles are safe when loaded with moderate concentrations of RA, without in vitro genotoxicity and cytotoxicity and with an in vivo safety profile in rats orally treated, thus opening new avenues for use in nutraceutical applications.
PMCID: PMC4977102  PMID: 27536103
solid lipid nanoparticles; rosmarinic acid; Witepsol and Carnauba waxes; safety profile; in vitro and animal toxicity
4.  The role of tissue engineering and biomaterials in cardiac regenerative medicine 
The Canadian journal of cardiology  2014;30(11):1307-1322.
In recent years, the development of three-dimensional engineered heart tissue (EHT) has made large strides forward due to advances in stem cell biology, materials science, pre-vascularization strategies and nanotechnology. As a result, the role of tissue engineering in cardiac regenerative medicine has become multi-faceted as new applications become feasible. Cardiac tissue engineering has long been established to have the potential to partially or fully restore cardiac function following cardiac injury. However, EHTs may also serve as surrogate human cardiac tissue for drug-related toxicity screening. Cardiotoxicity remains a major cause of drug withdrawal in the pharmaceutical industry. Unsafe drugs reach the market because pre-clinical evaluation is insufficient to weed out cardiotoxic drugs in all their forms. Bioengineering methods could provide functional and mature human myocardial tissues, i.e. physiologically relevant platforms, for screening the cardiotoxic effects of pharmaceutical agents and facilitate the discovery of new therapeutic agents. Finally, advances in induced pluripotent stem cells have made patient-specific EHTs possible, which opens up the possibility of personalized medicine. Herein, we give an overview of the present state of the art in cardiac tissue engineering, the challenges to the field and future perspectives.
PMCID: PMC4254531  PMID: 25442432
Engineered heart tissue; hPSC-derived cardiomyocytes; cardiac functional restoration; drug screening and discovery; personalized medicine
5.  Bioreactor for modulation of cardiac microtissue phenotype by combined static stretch and electrical stimulation 
Biofabrication  2014;6(2):024113.
We describe here a bioreactor capable of simultaneously applying mechanical and electrical field stimulation in conjunction with static strain and on-line force of contraction measurements. It consisted of a polydimethylsiloxane (PDMS) tissue chamber and a pneumatically driven stretch platform. The chamber contained eight tissue microwells (8.05 mm in length and 2.5 mm in width) with a pair of posts (2.78 mm in height and 0.8 mm in diameter) in each well to serve as fixation points and for measurements of contraction force. Carbon rods, stimulating electrodes, were placed into the PDMS chamber such that one pair stimulated four microwells. For feasibility studies, neonatal rat cardiomyocytes were seeded in collagen gels into the microwells. Following three days of gel compaction, electrical field stimulation at 3–4 V/cm and 1Hz, mechanical stimulation of 5% static strain or electromechanical stimulation (field stimulation at 3–4 V/cm, 1Hz and 5% static strain) were applied for 3 days. Cardiac microtissues subjected to electromechanical stimulation exhibited elevated amplitude of contraction and improved sarcomere structure as evidenced by sarcomeric α-actinin, actin and troponin T staining compared to microtissues subjected to electrical or mechanical stimulation alone or non-stimulated controls. The expression of atrial natriuretic factor and brain natriuretic peptide was also elevated in the electromechanically stimulated group.
PMCID: PMC4108215  PMID: 24876342
6.  Manipulating the Microvasculature and Its Microenvironment 
The microvasculature is a dynamic cellular system necessary for tissue health and function. Therapeutic strategies that target the microvasculature are expanding and evolving, including those promoting angiogenesis and microvascular expansion. When considering how to manipulate angiogenesis, either as part of a tissue construction approach or a therapy to improve tissue blood flow, it is important to know the microenvironmental factors that regulate and direct neovessel sprouting and growth. Much is known concerning both diffusible and matrix-bound angiogenic factors, which stimulate and guide angiogenic activity. How the other aspects of the extravascular microenvironment, including tissue biomechanics and structure, influence new vessel formation is less well known. Recent research, however, is providing new insights into these mechanisms and demonstrating that the extent and character of angiogenesis (and the resulting new microcirculation) is significantly affected. These observations and the resulting implications with respect to tissue construction and microvascular therapy are addressed.
PMCID: PMC4096003  PMID: 24580565
angiogenesis; microvessels; microvascular orientation; microvascular remodeling; microvessel guidance; three-dimensional (3D) vascular constructs; matrix mechanics
7.  Biowire: a new platform for maturation of human pluripotent stem cell derived cardiomyocytes 
Nature methods  2013;10(8):781-787.
Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSC) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here, we developed a new platform that combines three-dimensional cell cultivation in a microfabricated system with electrical stimulation to mature hPSC-derived cardiac tissues. We utilized quantitative structural, molecular and electrophysiological analyses to elucidate the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allowed for the generation of 3-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation markedly increased myofibril ultrastructural organization, displayed elevated conduction velocity and altered both the electrophysiological and Ca2+ handling properties versus non-stimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.
PMCID: PMC4071061  PMID: 23793239 CAMSID: cams3996
8.  Conversion to Sirolimus Ameliorates Cyclosporine-Induced Nephropathy in the Rat: Focus on Serum, Urine, Gene, and Protein Renal Expression Biomarkers 
BioMed Research International  2014;2014:576929.
Protocols of conversion from cyclosporin A (CsA) to sirolimus (SRL) have been widely used in immunotherapy after transplantation to prevent CsA-induced nephropathy, but the molecular mechanisms underlying these protocols remain nuclear. This study aimed to identify the molecular pathways and putative biomarkers of CsA-to-SRL conversion in a rat model. Four animal groups (n = 6) were tested during 9 weeks: control, CsA, SRL, and conversion (CsA for 3 weeks followed by SRL for 6 weeks). Classical and emergent serum, urinary, and kidney tissue (gene and protein expression) markers were assessed. Renal lesions were analyzed in hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome stains. SRL-treated rats presented proteinuria and NGAL (serum and urinary) as the best markers of renal impairment. Short CsA treatment presented slight or even absent kidney lesions and TGF-β, NF-κβ, mTOR, PCNA, TP53, KIM-1, and CTGF as relevant gene and protein changes. Prolonged CsA exposure aggravated renal damage, without clear changes on the traditional markers, but with changes in serums TGF-β and IL-7, TBARs clearance, and kidney TGF-β and mTOR. Conversion to SRL prevented CsA-induced renal damage evolution (absent/mild grade lesions), while NGAL (serum versus urine) seems to be a feasible biomarker of CsA replacement to SRL.
PMCID: PMC4055143  PMID: 24971338
9.  Topological and electrical control of cardiac differentiation and assembly 
Tissue engineering has developed many paradigms and techniques on how to best integrate cells and extracellular matrix to create in vitro structures that replicate native tissue. The strategy best suited for building these constructs depends mainly on the target cells, tissues, and organ of interest, and how readily their respective niches can be recapitulated in vitro with available technologies. In this review we examine engineered heart tissue and two techniques that can be used to induce tissue morphogenesis in artificial niches in vitro: engineered surface topology and electrical control of the system. For both the differentiation of stem cells into heart cells and further assembly of these cells into engineered tissues, these two techniques are effective in inducing in vivo like structure and function. Biophysical modulation through the control of topography and manipulation of the electrical microenvironment has been shown to have effects on cell growth and differentiation, expression of mature cardiac-related proteins and genes, cell alignment via cytoskeletal organization, and electrical and contractile properties. Lastly, we discuss the evolution and potential of these techniques, and bridges to regenerative therapies.
PMCID: PMC3706811  PMID: 23425700
10.  Dissecting the Role of Human Embryonic Stem Cell–Derived Mesenchymal Cells in Human Umbilical Vein Endothelial Cell Network Stabilization in Three-Dimensional Environments 
Tissue Engineering. Part A  2012;19(1-2):211-223.
The microvasculature is principally composed of two cell types: endothelium and mural support cells. Multiple sources are available for human endothelial cells (ECs) but sources for human microvascular mural cells (MCs) are limited. We derived multipotent mesenchymal progenitor cells from human embryonic stem cells (hES-MC) that can function as an MC and stabilize human EC networks in three-dimensional (3D) collagen-fibronectin culture by paracrine mechanisms. Here, we have investigated the basis for hES-MC-mediated stabilization and identified the pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) as a putative hES-MC-derived regulator of EC network stabilization in 3D in vitro culture. Pharmacological inhibition of the HGF receptor (Met) (1 μm SU11274) inhibits EC network formation in the presence of hES-MC. hES-MC produce and release HGF while human umbilical vein endothelial cells (HUVEC) do not. When HUVEC are cultured alone the networks collapse, but in the presence of recombinant human HGF or conditioned media from human HGF-transduced cells significantly more networks persist. In addition, HUVEC transduced to constitutively express human HGF also form stable networks by autocrine mechanisms. By enzyme-linked immunosorbent assay, the coculture media were enriched in both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), but at significantly different levels (Ang1=159±15 pg/mL vs. Ang2=30,867±2685 pg/mL) contributed by hES-MC and HUVEC, respectively. Although the coculture cells formed stabile network architectures, their morphology suggests the assembly of an immature plexus. When HUVEC and hES-MC were implanted subcutaneously in immune compromised Rag1 mice, hES-MC increased their contact with HUVEC along the axis of the vessel. This data suggests that HUVEC and hES-MC form an immature plexus mediated in part by HGF and angiopoietins that is capable of maturation under the correct environmental conditions (e.g., in vivo). Therefore, hES-MC can function as microvascular MCs and may be a useful cell source for testing EC–MC interactions.
PMCID: PMC3530951  PMID: 22971005
11.  Abnormal uterine bleeding as a presenting symptom is related to multiple uterine leiomyoma: an ultrasound-based study 
To determine the prevalence of uterine leiomyomas, diagnosed by ultrasound, in a private health care setting located in the central eastern region of Portugal, and to explore the demographic and clinical factors related to diagnosis and symptomatology.
Patients and methods
The files of 624 patients attending a private clinic in Covilhã, Portugal, from January 2 to December 31, 2010 were retrieved for evaluation. Pelvic ultrasound record, age, weight, height, age at menarche, number of pregnancies and deliveries, marital status, menstrual cycles characteristic, and contraceptive method at consultation were included in the analysis.
Uterine leiomyoma (UL) was diagnosed by ultrasonography in 161 (25.8%) patients. A single UL was diagnosed in 80 (49.7%) patients. In 79 (49.1%) patients, the largest leiomyoma had a dimension <20 mm. Prevalence of UL was age dependent: at 11.0% for women 20–39 years old; 45.4% for those aged 40–59 years; and 19.5% for women 60 years or older. Metrorrhagia was the most distressing presenting symptom. When menorrhagia was the presenting symptom, the probability of having an ultrasound diagnosis of UL was 73.3%. Metrorrhagia or menorrhagia, as presenting symptom, was significantly related to the ultrasound diagnosis of multiple ULs.
UL was especially prevalent in women aged between 40 and 59 years. Patients with multiple ULs had significantly more abnormal uterine bleeding. In patients with menorrhagia or metrorrhagia, special attention should be taken in searching for the presence of multiple ULs during ultrasound.
PMCID: PMC3814927  PMID: 24194648
fibroid; leiomyoma; prevalence; ultrasound; menorrhagia; metrorrhagia
12.  Early cardiac changes in a rat model of prediabetes: brain natriuretic peptide overexpression seems to be the best marker 
Diabetic cardiomyopathy (DCM) is defined as structural and functional changes in the myocardium due to metabolic and cellular abnormalities induced by diabetes mellitus (DM). The impact of prediabetic conditions on the cardiac tissue remains to be elucidated. The goal of this study was to elucidate whether cardiac dysfunction is already present in a state of prediabetes, in the presence of insulin resistance, and to unravel the underlying mechanisms, in a rat model without obesity and hypertension as confounding factors.
Two groups of 16-week-old Wistar rats were tested during a 9 week protocol: high sucrose (HSu) diet group (n = 7) – rats receiving 35% of sucrose in drinking water vs the vehicle control group (n = 7). The animal model was characterized in terms of body weight (BW) and the glycemic, insulinemic and lipidic profiles. The following parameters were assessed to evaluate possible early cardiac alterations and underlying mechanisms: blood pressure, heart rate, heart and left ventricle (LV) trophism indexes, as well as the serum and tissue protein and/or the mRNA expression of markers for fibrosis, hypertrophy, proliferation, apoptosis, angiogenesis, endothelial function, inflammation and oxidative stress.
The HSu-treated rats presented normal fasting plasma glucose (FPG) but impaired glucose tolerance (IGT), accompanied by hyperinsulinemia and insulin resistance (P < 0.01), confirming this rat model as prediabetic. Furthermore, although hypertriglyceridemia (P < 0.05) was observed, obesity and hypertension were absent. Regarding the impact of the HSu diet on the cardiac tissue, our results indicated that 9 weeks of treatment might be associated with initial cardiac changes, as suggested by the increased LV weight/BW ratio (P < 0.01) and a remarkable brain natriuretic peptide (BNP) mRNA overexpression (P < 0.01), together with a marked trend for an upregulation of other important mediators of fibrosis, hypertrophy, angiogenesis and endothelial lesions, as well as oxidative stress. The inflammatory and apoptotic markers measured were unchanged.
This animal model of prediabetes/insulin resistance could be an important tool to evaluate the early cardiac impact of dysmetabolism (hyperinsulinemia and impaired glucose tolerance with fasting normoglycemia), without confounding factors such as obesity and hypertension. Left ventricle hypertrophy is already present and brain natriuretic peptide seems to be the best early marker for this condition.
PMCID: PMC3599663  PMID: 23497124
Brain natriuretic peptide; Diabetic cardiomyopathy; Fibrosis; Hypertrophy; High-sucrose diet; Prediabetes
13.  Determinants of Microvascular Network Topologies in Implanted Neovasculatures 
During neovascularization, the end result is a new functional microcirculation comprised of a network of mature microvessels with specific topologies. While much is known concerning the mechanisms underlying the initiation of angiogenesis, it remains unclear how the final architecture of microcirculatory beds is regulated. To begin to address this, we determined the impact of angiogenic neovessel pre-patterning on the final microvascular network topology using an implant model of implant neovascularization.
Methods and Results
To test this, we used 3-D direct-write bioprinting or physical constraints in a manner permitting post-angiogenesis vascular remodeling and adaptation to pattern angiogenic microvascular precursors (neovessels formed from isolated microvessel segments) in 3-dimensional collagen gels prior to implantation and subsequent network formation. Neovasculatures pre-patterned into parallel arrays formed functional networks following 4 weeks post-implantation, but lost the pre-patterned architecture. However, maintenance of uniaxial physical constraints during post-angiogenesis remodeling of the implanted neovasculatures produced networks with aligned microvessels as well as an altered proportional distribution of arterioles, capillaries and venules.
Here we show that network topology resulting from implanted microvessel precursors is independent from pre-patterning of precursors but can be influenced by a patterning stimulus involving tissue deformation during post-angiogenesis remodeling and maturation.
PMCID: PMC3256738  PMID: 22053070
microcirculation; regeneration; bioprinting; vascular engineering; neovascularization
14.  Microvascular Mural Cell Functionality of Human Embryonic Stem Cell-Derived Mesenchymal Cells 
Tissue Engineering. Part A  2011;17(11-12):1537-1548.
Microvascular mural or perivascular cells are required for the stabilization and maturation of the remodeling vasculature. However, much less is known about their biology and function compared to large vessel smooth muscle cells. We have developed lines of multipotent mesenchymal cells from human embryonic stem cells (hES-MC); we hypothesize that these can function as perivascular mural cells. Here we show that the derived cells do not form teratomas in SCID mice and independently derived lines show similar patterns of gene expression by microarray analysis. When exposed to platelet-derived growth factor-BB, the platelet-derived growth factor receptor β is activated and hES-MC migrate in response to a gradient. We also show that in a serum-free medium, transforming growth factor β1 (TGFβ1) induces robust expression of multiple contractile proteins (α smooth muscle actin, smooth muscle myosin heavy chain, smooth muscle 22α, and calponin). TGFβ1 signaling is mediated through the TGFβR1/Alk5 pathway as demonstrated by inhibition of α smooth muscle actin expression by treatment of the Alk5-specific inhibitor SB525334 and stable retroviral expression of the Alk5 dominant negative (K232R). Coculture of human umbilical vein endothelial cell (HUVEC) with hES-MC maintains network integrity compared to HUVEC alone in three-dimensional collagen I-fibronectin by paracrine signaling. Using high-resolution laser confocal microscopy, we show that hES-MC also make direct contact with HUVEC. This demonstrates that hESC-derived mesenchymal cells possess the molecular machinery expected in a perivascular progenitor cells and can play a functional role in stabilizing EC networks in in vitro three-dimensional culture.
PMCID: PMC3098949  PMID: 21284534
15.  Vessel Arterial-Venous Plasticity in Adult Neovascularization 
PLoS ONE  2011;6(11):e27332.
Proper arterial and venous specification is a hallmark of functional vascular networks. While arterial-venous identity is genetically pre-determined during embryo development, it is unknown whether an analogous pre-specification occurs in adult neovascularization. Our goal is to determine whether vessel arterial-venous specification in adult neovascularization is pre-determined by the identity of the originating vessels.
Methods and Results
We assessed identity specification during neovascularization by implanting isolated microvessels of arterial identity from both mice and rats and assessing the identity outcomes of the resulting, newly formed vasculature. These microvessels of arterial identity spontaneously formed a stereotypical, perfused microcirculation comprised of the full complement of microvessel types intrinsic to a mature microvasculature. Changes in microvessel identity occurred during sprouting angiogenesis, with neovessels displaying an ambiguous arterial-venous phenotype associated with reduced EphrinB2 phosphorylation.
Our findings indicate that microvessel arterial-venous identity in adult neovascularization is not necessarily pre-determined and that adult microvessels display a considerable level of phenotypic plasticity during neovascularization. In addition, we show that vessels of arterial identity also hold the potential to undergo sprouting angiogenesis.
PMCID: PMC3221655  PMID: 22132096
16.  Angiogenic potential of microvessel fragments is independent of the tissue of origin and can be influenced by the cellular composition of the implants 
We have demonstrated that microvessel fragments (MFs) isolated from adipose retain angiogenic potential in vitro and form a mature, perfused network when implanted. However, adipose-derived microvessels are rich in pro-vascularizing cells that could uniquely drive neovascularization in adipose-derived MFs implants.
Investigate the ability of microvessel fragments from a different vascular bed to recapitulate adipose-derived microvessel angiogenesis and network formation and analyze adipose-derived vessel plasticity by assessing whether vessel function could be modulated by astrocyte-like cells.
MFs were isolated by limited collagenase digestion from rodent brain or adipose and assembled into 3D collagen gels in the presence or absence of GRPs. The resulting neovasculatures that formed following implantation were assessed by measuring 3-D vascularity and vessel permeability to small and large molecular tracers.
Similar to adipose-derived MFs, brain-derived MFs can sprout and form a perfused neovascular network when implanted. Furthermore, when co-implanted in the constructs, GRPs caused adipose-derived vessels to express the brain endothelial marker glucose transporter-1 and to significantly reduce microvessel permeability.
Neovascularization involving isolated microvessel elements is independent of the tissue origin and degree of vessel specialization. In addition, adipose-derived vessels have the ability to respond to environmental signals and change vessel characteristics.
PMCID: PMC3057771  PMID: 21040121
Angiogenesis; vessel permeability; glial restricted precursors; astrocytes; angiogenesis assay
17.  Angiogenesis in a Microvascular Construct for Transplantation Depends on the Method of Chamber Circulation 
Tissue Engineering. Part A  2009;16(3):795-805.
Effective tissue prevascularization depends on new vessel growth and subsequent progression of neovessels into a stable microcirculation. Isolated microvessel fragments in a collagen-based microvascular construct (MVC) spontaneously undergo angiogenesis in static conditions in vitro but form a new microcirculation only when implanted in vivo. We have designed a bioreactor, the dynamic in vitro perfusion (DIP) chamber, to culture MVCs in vitro with perfusion. By altering bioreactor circulation, microvessel fragments in the DIP chamber either maintained stable, nonsprouting, patent vessel morphologies or sprouted endothelial neovessels that extended out into the surrounding collagen matrix (i.e., angiogenesis), yielding networks of neovessels within the MVC. Neovessels formed in regions of the construct predicted by simulation models to have the steepest gradients in oxygen levels and expressed hypoxia inducible factor-1α. By altering circulation conditions in the DIP chamber, we can control, possibly by modulating hypoxic stress, prevascularizing activity in vitro.
PMCID: PMC2862615  PMID: 19778185
18.  Preventive but Not Curative Efficacy of Celecoxib on Bladder Carcinogenesis in a Rat Model 
Mediators of Inflammation  2011;2010:380937.
To evaluate the effect of a cyclooxygenase 2 inhibitor, celecoxib (CEL), on bladder cancer inhibition in a rat model, when used as preventive versus as curative treatment. The study comprised 52 male Wistar rats, divided in 5 groups, during a 20-week protocol: control: vehicle, carcinogen: 0.05% of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), CEL: 10 mg/kg/day of the selective COX-2 inhibitor Celebrex, preventive CEL (CEL+BBN-P), and curative CEL (BBN+CEL-C) groups. Although tumor growth was markedly inhibited by the preventive application of CEL, it was even aggravated by the curative treatment. The incidence of gross bladder carcinoma was: control 0/8(0%), BBN 13/20(65%), CEL 0/8(0%), CEL+BBN-P 1/8(12.5%), and BBN+CEL-C 6/8(75%). The number and volume of carcinomas were significantly lower in the CEL+BBN-P versus BBN, accompanied by an ample reduction in hyperplasia, dysplasia, and papillary tumors as well as COX-2 immunostaining. In spite of the reduction of tumor volumes in the curative BBN+CEL-C group, tumor malignancy was augmented. An anti-inflammatory and antioxidant profile was encountered only in the group under preventive treatment. In conclusion, preventive, but not curative, celecoxib treatment promoted a striking inhibitory effect on bladder cancer development, reinforcing the potential role of chemopreventive strategies based on cyclooxygenase 2 inhibition.
PMCID: PMC3042634  PMID: 21403827
19.  Regular physical exercise training assists in preventing type 2 diabetes development: focus on its antioxidant and anti-inflammatory properties 
Diabetes mellitus has emerged as one of the main alarms to human health in the 21st century. Pronounced changes in the human environment, behavior and lifestyle have accompanied globalization, which resulted in escalating rates of both obesity and diabetes, already described as diabesity. This pandemic causes deterioration of life quality with high socio-economic costs, particularly due to premature morbidity and mortality. To avoid late complications of type 2 diabetes and related costs, primary prevention and early treatment are therefore necessary. In this context, effective non-pharmacological measures, such as regular physical activity, are imperative to avoid complications, as well as polymedication, which is associated with serious side-effects and drug-to-drug interactions.
Our previous work showed, in an animal model of obese type 2 diabetes, the Zucker Diabetic Fatty (ZDF) rat, that regular and moderate intensity physical exercise (training) is able, per se, to attenuate insulin resistance and control glycaemia, dyslipidaemia and blood pressure, thus reducing cardiovascular risk, by interfering with the pathophysiological mechanisms at different levels, including oxidative stress and low-grade inflammation, which are key features of diabesity.
This paper briefly reviews the wide pathophysiological pathways associated with Type 2 diabetes and then discusses in detail the benefits of training therapy on glycaemic control and on cardiovascular risk profile in Type 2 diabetes, focusing particularly on antioxidant and anti-inflammatory properties. Based on the current knowledge, including our own findings using an animal model, it is concluded that regular and moderate intensity physical exercise (training), due to its pleiotropic effects, could replace, or at least reduce, the use of anti-diabetic drugs, as well as of other drugs given for the control of cardiovascular risk factors in obese type 2 diabetic patients, working as a physiological "polypill".
PMCID: PMC3041659  PMID: 21276212
20.  Implanted Microvessels Progress through Distinct Neovascularization Phenotypes 
Microvascular research  2009;79(1):10.
We have previously demonstrated that implanted microvessels form a new microcirculation with minimal host-derived vessel investment. Our objective was to define the vascular phenotypes present during neovascularization in these implants and identify post-angiogenesis events. Morphological, functional and transcriptional assessments identified three distinct vascular phenotypes in the implants: sprouting angiogenesis, neovascular remodeling, and network maturation. A sprouting angiogenic phenotype appeared first, characterized by high proliferation and low mural cell coverage. This was followed by a neovascular remodeling phenotype characterized by a perfused, poorly organized neovascular network, reduced proliferation, and re-associated mural cells. The last phenotype included a vascular network organized into a stereotypical tree structure containing vessels with normal perivascular cell associations. In addition, proliferation was low and was restricted to the walls of larger microvessels. The transition from angiogenesis to neovascular remodeling coincided with the appearance of blood flow in the implant neovasculature. Analysis of vascular-specific and global gene expression indicates that the intermediate, neovascular remodeling phenotype is transcriptionally distinct from the other two phenotypes. Therefore, this vascular phenotype likely is not simply a transitional phenotype but a distinct vascular phenotype involving unique cellular and vascular processes. Furthermore, this neovascular remodeling phase may be a normal aspect of the general neovascularization process. Given that this phenotype is arguably dysfunctional, many of the microvasculatures present within compromised or diseased tissues may not represent a failure to progress appropriately through a normally occurring neovascularization phenotype.
PMCID: PMC2813398  PMID: 19833141
neovascularization; neovessel; sprouting; angiogenesis; microvascular remodeling; microcirculation; structural adaptation; gene expression
21.  Effects of Sitagliptin Treatment on Dysmetabolism, Inflammation, and Oxidative Stress in an Animal Model of Type 2 Diabetes (ZDF Rat) 
Mediators of Inflammation  2010;2010:592760.
The purpose of this paper is to evaluate the chronic effect of sitagliptin on metabolic profile, inflammation, and redox status in the Zucker Diabetic Fatty (ZDF) rat, an animal model of obese type 2 diabetes. Diabetic and obese ZDF (fa/fa) rats and their controls (ZDF +/+) were treated during 6 weeks with vehicle (control) and sitagliptin (10 mg/kg/bw). Glucose, HbA1c, insulin, Total-c, TGs, IL-1β, TNF-α, CRPhs, and adiponectin were assessed in serum and MDA and TAS in serum, pancreas, and heart. Pancreatic histology was also evaluated. Sitagliptin in diabetic rats promoted a decrease in glucose, HbA1c, Total-c, and TGs accompanied by a partial prevention of insulinopenia, together, with a decrease in CRPhs and IL-1β. Sitagliptin also showed a positive impact on lipid peroxidation and hypertension prevention. In conclusion, chronic sitagliptin treatment corrected the glycaemic dysmetabolism, hypertriglyceridaemia, inflammation, and hypertension, reduced the severity of the histopathological lesions of pancreatic endocrine and exocrine tissues, together with a favourable redox status, which might be a further advantage in the management of diabetes and its proatherogenic comorbidities.
PMCID: PMC2905949  PMID: 20652060

Results 1-21 (21)