Polymer-based scaffold designs for in situ vascular tissue engineering: controlling recruitment and differentiation behavior of endothelial colony forming cells

In situ vascular tissue engineering has been proposed as a promising approach to fulfill the need for small-diameter blood vessel substitutes. The approach comprises the use of a cell-free instructive scaffold to guide and control cell recruitment, differentiation, and tissue formation at the locus of implantation. Here we review the design parameters for such scaffolds, with special emphasis on differentiation of recruited ECFCs into the different lineages that constitute the vessel wall. Next to defining the target properties of the vessel, we concentrate on the target cell source, the ECFCs, and on the environmental control of the fate of these cells within the scaffold. The prospects of the approach are discussed in the light of current technical and biological hurdles.     

Testing Foundations of Biological Scaling Theory Using Automated Measurements of Vascular Networks

Scientists have long sought to understand how vascular networks supply blood and oxygen to cells throughout the body. Recent work focuses on principles that constrain how vessel size changes through branching generations from the aorta to capillaries and uses scaling exponents to quantify these changes. Prominent scaling theories predict that combinations of these exponents explain how metabolic, growth, and other biological rates vary with body size. Nevertheless, direct measurements of individual vessel segments have been limited because existing techniques for measuring vasculature are invasive, time consuming, and technically difficult. We developed software that extracts the length, radius, and connectivity of in vivo vessels from contrast-enhanced 3D Magnetic Resonance Angiography. Using data from 20 human subjects, we calculated scaling exponents by four methods—two derived from local properties of branching junctions and two from whole-network properties. Although these methods are often used interchangeably in the literature, we do not find general agreement between these methods, particularly for vessel lengths. Measurements for length of vessels also diverge from theoretical values, but those for radius show stronger agreement. Our results demonstrate that vascular network models cannot ignore certain complexities of real vascular systems and indicate the need to discover new principles regarding vessel lengths.     

A new fundamental bioheat equation for muscle tissue--part II: Temperature of SAV vessels

In this study, a new theoretical framework was developed to investigate temperature variations along countercurrent SAV blood vessels from 300 to 1000 microm diameter in skeletal muscle. Vessels of this size lie outside the range of validity of the Weinbaum-Jiji bioheat equation and, heretofore, have been treated using discrete numerical methods. A new tissue cylinder surrounding these vessel pairs is defined based on vascular anatomy, Murray's law, and the assumption of uniform perfusion. The thermal interaction between the blood vessel pair and surrounding tissue is investigated for two vascular branching patterns, pure branching and pure perfusion. It is shown that temperature variations along these large vessel pairs strongly depend on the branching pattern and the local blood perfusion rate. The arterial supply temperature in different vessel generations was evaluated to estimate the arterial inlet temperature in the modified perfusion source term for the s vessels in Part I of this study. In addition, results from the current research enable one to explore the relative contribution of the SAV vessels and the s vessels to the overall thermal equilibration between blood and tissue.     

Angiogenesis and vascular remodelling in normal and cancerous tissues

Vascular development and homeostasis are underpinned by two fundamental features: the generation of new vessels to meet the metabolic demands of under-perfused regions and the elimination of vessels that do not sustain flow. In this paper we develop the first multiscale model of vascular tissue growth that combines blood flow, angiogenesis, vascular remodelling and the subcellular and tissue scale dynamics of multiple cell populations. Simulations show that vessel pruning, due to low wall shear stress, is highly sensitive to the pressure drop across a vascular network, the degree of pruning increasing as the pressure drop increases. In the model, low tissue oxygen levels alter the internal dynamics of normal cells, causing them to release vascular endothelial growth factor (VEGF), which stimulates angiogenic sprouting. Consequently, the level of blood oxygenation regulates the extent of angiogenesis, with higher oxygenation leading to fewer vessels. Simulations show that network remodelling (and de novo network formation) is best achieved via an appropriate balance between pruning and angiogenesis. An important factor is the strength of endothelial tip cell chemotaxis in response to VEGF. When a cluster of tumour cells is introduced into normal tissue, as the tumour grows hypoxic regions form, producing high levels of VEGF that stimulate angiogenesis and cause the vascular density to exceed that for normal tissue. If the original vessel network is sufficiently sparse then the tumour may remain localised near its parent vessel until new vessels bridge the gap to an adjacent vessel. This can lead to metastable periods, during which the tumour burden is approximately constant, followed by periods of rapid growth.     

Netrin-4 acts as a pro-angiogenic factor during zebrafish development

Netrins form a heterogeneous family of laminin-related molecules with multifunctional activities. Netrin-4, the most distant member of this family, is related to the laminin β chain and has recently been proposed to play an important role in embryonic and pathological angiogenesis. However, the data reported so far lead to the apparently contradictory conclusions supporting Netrin-4 as either a pro- or an anti-angiogenic factor. To elucidate this controversy, Netrin-4 was analyzed for a vascular activity in both cell-based models (human umbilical vein endothelial cells and human umbilical artery endothelial cells) and two zebrafish models: the wild-type AB/Tü strain and the transgenic Tg(fli1a:EGFP)(y1) strain. We show that Netrin-4 is expressed in endothelial cells and in the zebrafish vascular system. We also show evidence that Netrin-4 activates various kinases and induces various biological effects directly linked to angiogenesis in vitro. Using a morpholinos strategy, we demonstrate that Netrin-4 expression is crucial for zebrafish vessel formation and that a blood vessel formation defect induced by netrin-4 morpholinos can be partially rescued through drug delivery leading to protein kinase activation. Together these data underscore the crucial role of Netrin-4 in blood vessel formation and the involvement of protein kinases activation in Netrin-4-induced biological effects related to vascular development.     

Cryopreservation of vascular tissues

Cryopreservation of human blood vessels may become an important tool in bypass surgery and peripheral vascular reconstruction. Ideally cryopreservation of a blood vessel should preserve functional characteristics comparable to those of fresh controls. The key advantage of cryopreservation is the fact that storage at deep subzero temperatures allows storage of structurally intact living vascular tissues for virtually infinite time. Originally developed for long-time storage of isolated cells, the techniques of cryopreservation of tissues are challenged by the fact that these are complex multicellular systems containing diverse types of cells with differing requirements for optimal preservation. Therefore, the post-thaw functional activity of vascular tissues is determined by the type of blood vessel and, in addition, by the cell packing effect. Moreover, evidence from pharmacological studies suggests that cryopreservation induces tissue specific changes in transmembrane signaling and the mechanisms coupling intracellular calcium release, sensitivity and calcium entry into the smooth muscle cells.     

Immunohistochemical investigation of lymphatic vessel formation control in mouse tooth development: lymphatic vessel-forming factors and receptors in tooth development in mice

The presence of lymphatic vessels in dental pulp has recently been controversial, and no conclusion has been reached. In this study, we investigated the control of lymphangiogenesis with dental pulp development in the mouse mandibular molar using VEGF-C, VEGF-D, and VEGFR-3 as indices of lymphatic vessel-controlling factors. In addition, to distinguish blood and lymphatic vascular epithelial cells, we performed immunohistochemical analysis using von Willebrand factor (vWF) and statistical analysis. In dental papilla in the bell-stage non-calcified period, mesenchymal cells positive for VEGF-C, VEGF-D, and VEGFR-3 increased and lumen-forming endothelial cells were noted, but vWF was negative, suggesting that these were actively forming lymphatic vessels. Positive undifferentiated mesenchymal cells, an increase in endothelial cells in dental pulp, and lumen expansion were noted early after birth. Positivity was also detected in the odontoblast layer and sheath of Hertwig after birth, suggesting that these factors also play important roles in odontoblast differentiation and maturation and periodontal ligament and tooth root formation. We embryologically clarified lymphatic vessel formation in dental pulp and a process of lymphatic vessel formation from blood vessels, suggesting involvement of the surrounding tissue, odontoblasts, and sheath of Hertwig in vessel formation.     

Cryopreservation of vascular tissues

Cryopreservation of human blood vessels may become an important tool in bypass surgery and peripheral vascular reconstruction. Ideally cryopreservation of a blood vessel should preserve functional characteristics comparable to those of fresh controls. The key advantage of cryopreservation is the fact that storage at deep subzero temperatures allows storage of structurally intact living vascular tissues for virtually infinite time. Originally developed for long-time storage of isolated cells, the techniques of cryopreservation of tissues are challenged by the fact that these are complex multicellular systems containing diverse types of cells with differing requirements for optimal preservation. Therefore, the post-thaw functional activity of vascular tissues is determined by the type of blood vessel and, in addition, by the cell packing effect. Moreover, evidence from pharmacological studies suggests that cryopreservation induces tissue specific changes in transmembrane signaling and the mechanisms coupling intracellular calcium release, sensitivity and calcium entry into the smooth muscle cells.     

Blood Flow Changes Coincide with Cellular Rearrangements during Blood Vessel Pruning in Zebrafish Embryos

After the initial formation of a highly branched vascular plexus, blood vessel pruning generates a hierarchically structured network with improved flow characteristics. We report here on the cellular events that occur during the pruning of a defined blood vessel in the eye of developing zebrafish embryos. Time-lapse imaging reveals that the connection of a new blood vessel sprout with a previously perfused multicellular endothelial tube leads to the formation of a branched, Y-shaped structure. Subsequently, endothelial cells in parts of the previously perfused branch rearrange from a multicellular into a unicellular tube, followed by blood vessel detachment. This process is accompanied by endothelial cell death. Finally, we show that differences in blood flow between neighboring vessels are important for the completion of the pruning process. Our data suggest that flow induced changes in tubular architecture ensure proper blood vessel pruning.     

Vascular wall extracellular matrix proteins and vascular diseases

Extracellular matrix proteins form the basic structure of blood vessels. Along with providing basic structural support to blood vessels, matrix proteins interact with different sets of vascular cells via cell surface integrin or non-integrin receptors. Such interactions induce vascular cell de novo synthesis of new matrix proteins during blood vessel development or remodeling. Under pathological conditions, vascular matrix proteins undergo proteolytic processing, yielding bioactive fragments to influence vascular wall matrix remodeling. Vascular cells also produce alternatively spliced variants that induce vascular cell production of different matrix proteins to interrupt matrix homeostasis, leading to increased blood vessel stiffness; vascular cell migration, proliferation, or death; or vascular wall leakage and rupture. Destruction of vascular matrix proteins leads to vascular cell or blood-borne leukocyte accumulation, proliferation, and neointima formation within the vascular wall; blood vessels prone to uncontrolled enlargement during blood flow diastole; tortuous vein development; and neovascularization from existing pathological tissue microvessels. Here we summarize discoveries related to blood vessel matrix proteins within the past decade from basic and clinical studies in humans and animals — from expression to cross-linking, assembly, and degradation under physiological and vascular pathological conditions, including atherosclerosis, aortic aneurysms, varicose veins, and hypertension.     

Tracking Dynamic Microvascular Changes during Healing after Complete Biopsy Punch on the Mouse Pinna Using Optical Microangiography

Optical microangiography (OMAG) and Doppler optical microangiography (DOMAG) are two non-invasive techniques capable of determining the tissue microstructural content, microvasculature angiography, and blood flow velocity and direction. These techniques were used to visualize the acute and chronic microvascular and tissue responses upon an injury in vivo. A tissue wound was induced using a 0.5 mm biopsy punch on a mouse pinna. The changes in the microangiography, blood flow velocity and direction were quantified for the acute (<30 min) wound response and the changes in the tissue structure and microangiography were determined for the chronic wound response (30 min–60 days). The initial wound triggered recruitment of peripheral capillaries, as well as redirection of main arterial and venous blood flow within 3 min. The complex vascular networks and new vessel formation were quantified during the chronic response using fractal dimension. The highest rate of wound closure occurred between days 8 and 22. The vessel tortuosity increased during this time suggesting angiogenesis. Taken together, these data signify that OMAG has the capability to track acute and chronic changes in blood flow, microangiography and structure during wound healing. The use of OMAG has great potential to improve our understanding of vascular and tissue responses to injury in order to develop more effective therapeutics.     

Role of reactive oxygen and nitrogen species in the vascular responses to inflammation

Inflammation is a complex and potentially life-threatening condition that involves the participation of a variety of chemical mediators, signaling pathways, and cell types. The microcirculation, which is critical for the initiation and perpetuation of an inflammatory response, exhibits several characteristic functional and structural changes in response to inflammation. These include vasomotor dysfunction (impaired vessel dilation and constriction), the adhesion and transendothelial migration of leukocytes, endothelial barrier dysfunction (increased vascular permeability), blood vessel proliferation (angiogenesis), and enhanced thrombus formation. These diverse responses of the microvasculature largely reflect the endothelial cell dysfunction that accompanies inflammation and the central role of these cells in modulating processes as varied as blood flow regulation, angiogenesis, and thrombogenesis. The importance of endothelial cells in inflammation-induced vascular dysfunction is also predicated on the ability of these cells to produce and respond to reactive oxygen and nitrogen species. Inflammation seems to upset the balance between nitric oxide and superoxide within (and surrounding) endothelial cells, which is necessary for normal vessel function. This review is focused on defining the molecular targets in the vessel wall that interact with reactive oxygen species and nitric oxide to produce the characteristic functional and structural changes that occur in response to inflammation. This analysis of the literature is consistent with the view that reactive oxygen and nitrogen species contribute significantly to the diverse vascular responses in inflammation and supports efforts that are directed at targeting these highly reactive species to maintain normal vascular health in pathological conditions that are associated with acute or chronic inflammation.     

Endothelial progenitor cells: A source for therapeutic vasculogenesis?

Angiogenesis has been defined as sprouting of blood vessels from pre-existing vascular structures. Risau and co-workers defined the term vasculogenesis while studying the formation of new blood vessels in embryoid bodies. This process is characterized by the recruitment of endothelial progenitor cells (EPC) to sites of new vessel formation with subsequent differentiation of EPC into mature endothelial cells, extensively proliferating in situ. Data from recent years provided evidence that EPC also exist in the adult and contribute to new vessel formation, a process called post-natal vasculogenesis. The existence of EPC has been convincingly shown in both, animals and humans. They represent a perfect cellular progenitor cell population for the ex vivo generation of EC, which in turn serve as cellular source for therapeutic vasculogenesis or tumor targeting. This review provides an overview on this hot topic of cellular-based therapeutic concepts and the therapeutic potential of ex vivo generated EPC.     

The delay phenomenon: the story unfolds

Our previous studies have shown that when a flap is delayed, there is dilation of existing vessels within the flap not ingrowth of new vessels. The maximal anatomic effect on the arterial tree occurs at the level of the reduced-caliber "choke" anastomotic vessels that link adjacent vascular territories. To further investigate the sequence of anatomic changes that occurs during the delay phenomenon, a large series of 200 rabbits and 17 dogs underwent a flap delay procedure in either skin or muscle and the tissues were examined at postoperative periods between 1 hour and 1 year by using well-established fluorescein, angiographic, light microscopic, immunohistochemical, and electron microscopic techniques. These data in the rabbit skin consistently demonstrated an initial period of vasoconstriction that resolved within 3 hours postoperatively and was followed by an active and progressive dilation of choke vessels that was most dramatic between 48 and 72 hours. In vivo intravenous fluorescein dye testing revealed an interesting parallel in that there was a temporary barrier to the flow of fluorescein that occurred at the level of the choke vessels immediately after the flap was raised and that this temporary barrier-continued to impede the flow toward the flap tip in rabbits where flaps had been delayed for periods up to 72 hours. Thereafter, there was no obstruction to the flow of fluorescein along the flap. During this early delay period of 3 days, light microscopy revealed a decrease in vessel wall thickness associated with an increase in lumen diameter. Over the next 4 days, the luminal diameter continued to dilate to a lesser extent and the vessel wall thickened. Immunohistochemical analysis showed increased cell division, maximal between 24 and 72 hours, in all layers of the choke vessel wall. During this same postoperative interval, transmission electron microscopy revealed phenotypic changes in smooth muscle cells from contractile to synthetic cells. Hypertrophy of the smooth muscle cells was also observed. The vascular endothelium, which initially showed evidence of denudation, was restored to a healthy intact appearance within the first week after delay. When followed for longer periods, long-term studies of the delayed flap of up to 1 year demonstrated dramatically a permanent dilation of the choke vessel lumen and a thickening of the choke vessel wall. In canine studies, one rectus abdominis muscle was delayed by ligating the deep inferior epigastric artery. The time sequence of choke vessel dilation, studied by sequential angiograms in vivo, was comparable to that of the rabbit skin model. To ascertain the permanence and irreversibility of this dilation, the normal circulation of the delayed rectus abdominis muscle was restored by reanastomosing the deep inferior epigastric artery. Even after a recovery period of up to 3 months, the choke vessels remained dilated and tortuous instead of reverting to their original narrow diameters. From this work, it is suggested that the choke vessel dilation seen in the delay period is a permanent and irreversible event. It is an active process associated with both an increase (hyperplasia) and an enlargement (hypertrophy) of the cells in all layers of the choke artery wall and a resultant increase in caliber of these vessels. The time sequence for delay appears to be similar in different species and in different tissues, suggesting the possibility of a universal process for delay.     

Embryonic development of the proepicardium and coronary vessels

In the last few years, an increasing interest in progenitor cells has been noted. These cells are a source of undifferentiated elements from which cellular components of tissues and organs develop. Such progenitor tissue delivering stem cells for cardiac development is the proepicardium. The proepicardium is a transient organ which occurs near the venous pole of the embryonic heart and protrudes to the pericardial cavity. The proepicardium is a source of the epicardial epithelium delivering cellular components of vascular wall and interstitial tissue fibroblasts. It contributes partially to a fibrous tissue skeleton of the heart. Epicardial derived cells play also an inductive role in differentiation of cardiac myocytes into conductive tissue of the heart. Coronary vessel formation proceeds by vasculogenesis and angiogenesis. The first tubules are formed from blood islands which subsequently coalesce forming the primitive vascular plexus. Coronary arteries are formed by directional growth of vascular protrusions towards the aorta and establishing contact with the aortic wall. The coronary vascular wall matures by attaching smooth muscle cell precursors and fibroblast precursors to the endothelial cell wall. The cells of tunica media differentiate subsequently into vascular smooth muscle by acquiring specific contractile and cytoskeletal markers of smooth muscle cells in a proximal - distal direction. The coronary artery wall matures first before cardiac veins. Maturity of the vessel wall is demonstrated by the specific shape of the internal surface of the vascular wall.     

Mathematical Modelling of a Brain Tumour Initiation and Early Development: A Coupled Model of Glioblastoma Growth,Pre-Existing Vessel Co-Option,Angiogenesis and Blood Perfusion

We propose a coupled mathematical modelling system to investigate glioblastoma growth in response to dynamic changes in chemical and haemodynamic microenvironments caused by pre-existing vessel co-option, remodelling, collapse and angiogenesis. A typical tree-like architecture network with different orders for vessel diameter is designed to model pre-existing vasculature in host tissue. The chemical substances including oxygen, vascular endothelial growth factor, extra-cellular matrix and matrix degradation enzymes are calculated based on the haemodynamic environment which is obtained by coupled modelling of intravascular blood flow with interstitial fluid flow. The haemodynamic changes, including vessel diameter and permeability, are introduced to reflect a series of pathological characteristics of abnormal tumour vessels including vessel dilation, leakage, angiogenesis, regression and collapse. Migrating cells are included as a new phenotype to describe the migration behaviour of malignant tumour cells. The simulation focuses on the avascular phase of tumour development and stops at an early phase of angiogenesis. The model is able to demonstrate the main features of glioblastoma growth in this phase such as the formation of pseudopalisades, cell migration along the host vessels, the pre-existing vasculature co-option, angiogenesis and remodelling. The model also enables us to examine the influence of initial conditions and local environment on the early phase of glioblastoma growth.     

Tubedown-1 in remodeling of the developing vitreal vasculature in vivo and regulation of capillary outgrowth in vitro

Tubedown-1 (tbdn-1) is a mammalian homologue of the N-terminal acetyltransferase subunit NAT1 of Saccharomyces cerevisiae and copurifies with an acetyltransferase activity. Tbdn-1 expression in endothelial cells becomes downregulated during the formation of capillary-like structures in vitro and is regulated in vivo in a manner which suggests a functional role in dampening blood vessel development. Here we show that tbdn-1 is expressed highly in the vitreal vascular network (tunica vasculosa lentis and vasa hyaloidea propria) during the pruning and remodeling phases of this transient structure. The vitreal blood vessels of mice harboring a targeted inactivation of TGF-beta2 fail to remodel and abnormally accumulate, a phenomenon reminiscent of the ocular pathology resembling persistent fetal vasculature (PFV) in humans. Since suppression of normal tbdn-1 expression has been previously observed in retinal vessel proliferation, we analyzed vitreal vascular changes and tbdn-1 expression in TGF-beta2(-/-) eyes. The nuclei of vitreal vessel endothelial cells in TGF-beta2(-/-) eyes express proliferating cell nuclear antigen (PCNA) and exhibit increased levels of active (P42/44)mitogen-activated protein kinase (phospho-(P42/44)MAPK), characteristics consistent with proliferative endothelial cells. In contrast to normal vitreal vessels, collagen IV expression exhibited a disorganized pattern in the TGF-beta2(-/-) vitreal vessels, suggesting vessel disorganization and possibly a breakdown of vessel basal laminae. Moreover, vitreal vessels of TGF-beta2(-/-) mice lack expression of pericyte markers (CD13, alpha smooth muscle actin) and show ultrastructural changes consistent with pericyte degeneration. The accumulating vitreal blood vessels of TGF-beta2(-/-) mice, while maintaining expression of the endothelial marker von Willebrand Factor, show a significant decrease in the expression of tbdn-1. We addressed the functional role of tbdn-1 in the regulation of vitreal blood vessels using an in vitro model of choroid-retina capillary outgrowth. Clones of the RF/6A fetal choroid-retina endothelial cell line showing suppression of tbdn-1 levels after overexpression of an antisense TBDN-1 cDNA display a significant increase in the formation of capillary-like structures in vitro compared with controls. These findings suggest that tbdn-1 inhibits capillary-like formation in vitro and may serve to dampen vitreal blood vessel formation preceding the regression of the vitreal vasculature during development. Our results also suggest that tbdn-1 may participate with TGF-beta2 in regulating normal development of the vitreal vasculature.     

First trimester trophoblasts forming endothelial-like tubes in vitro emulate a ‘blood vessel development’ gene expression profile

Extravillous cytotrophoblasts isolated from first trimester placenta, and immortalised cell lines derived from them, have the intrinsic ability to form endothelial-like tubes when cultured on Matrigel™ extracellular matrix. This in vitro tube formation may model placental angiogenesis and/or endovascular differentiation by trophoblasts. To interpret the relevance of this phenomenon to placental development, we used a gene expression microarray approach to identify which genes and pathways are associated with the tube-forming phenotype of HTR8/SVneo first trimester trophoblasts (HTR8-M), compared with HTR8/SVneo not forming tubes on plastic culture surface (HTR8-P). Furthermore, we used weighted gene co-expression network analysis (WGCNA) of microarray data to identify modules of co-expressed genes underlying the biological processes. There were 481 genes differentially expressed between HTR8-M and HTR8-P and these were significantly enriched for blood vessel development and related gene ontologies. WGCNA clustered the genes into 9 co-expression modules. One module was significantly associated with HTR8-M (p = 1.15E-05) and contained genes involved in actin cytoskeleton organization, cell migration and blood vessel development, consistent with tube formation on Matrigel. Another module was significantly associated with HTR8-P (p = 1.94E-05) and was enriched for genes involved in mitosis, consistent with proliferation by cells on plastic which do not differentiate. Up-regulation of angiogenesis and vascular development pathways in endovascular trophoblasts in vivo could underpin spiral artery remodelling processes, which are defective in preeclamptic pregnancies.     

Endothelial changes and microvascular leakage due to hyperthermia in chick embryos

The effects of hyperthermia on the developing 2- and 3-day chick embryo were studied by vital microscopy, in vivo microangiography and electron microscopy of post-capillary venules of the pellucid area of the yolk sac. Hyperthermia of 3 degrees C and 4 degrees C produced significant microvascular changes and perivascular oedema. The microvascular defects were characterized by interruption of the endothelial lining and the presence of blood cells breaking through the vessel walls. In addition, there were numerous inter-endothelial gaps with wide subendothelial spaces. Microangiography showed leakage from the vessel walls. It is concluded that hyperthermia produces vessel wall injury and induces the formation of gaps between endothelial cells resulting in extravasation of plasma and blood cells. These gaps are similar to those produced by biochemical mediators of inflammation. It is suggested that these microvascular changes with pathological leakage may play important roles in abnormal vascular and embryonic development.