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.     

A systems biology view of blood vessel growth and remodelling

Blood travels throughout the body in an extensive network of vessels – arteries, veins and capillaries. This vascular network is not static, but instead dynamically remodels in response to stimuli from cells in the nearby tissue. In particular, the smallest vessels – arterioles, venules and capillaries – can be extended, expanded or pruned, in response to exercise, ischaemic events, pharmacological interventions, or other physiological and pathophysiological events. In this review, we describe the multi‐step morphogenic process of angiogenesis – the sprouting of new blood vessels – and the stability of vascular networks in vivo. In particular, we review the known interactions between endothelial cells and the various blood cells and plasma components they convey. We describe progress that has been made in applying computational modelling, quantitative biology and high‐throughput experimentation to the angiogenesis process.     

Analysis of the structural parameters of the blood flow pathways in the microcirculatory system]

The reconstruction of the mesenterium microcirculatory bed was performed intravitally in albino rats and cats after biomicrophotograms. The number, length and caliber of arterioles, pericapillary arteriolec, capillaries, postcapillary venules and venules of the mesenterium were measured. According to these data summary indices of the cross section, surface and volume of the vessels of various functional subdivisions of the microcirculatory bed were calculated. The blood volume entering the microcirculatory system of the albino rat's mesenterium is distributed in the vessels as follows: 8,4% -- arterioles, 10,2% -- pericapillary arterioles, 41,9% -- capillaries, 22,1% -- postcapillary venules and 17,4% -- venules. Similar correlations were found in the cat. The working surface of capillaries is 60--70% of the working surface of all the vessels of the mesenterial microcirculatory system. The evidence of the functional variability of the microcirculatory bed geometry depending on the tissue needs in blood supply is presented.     

Condition of the microcirculatory bed of the bulbar conjunctiva in physiological and pathological pregnancies

By means of biomicroscopic method the bulbar conjunctiva in 150 women (18-35 years of age) have been examined. Of them: 30 healthy women make the control group; the second group includes 60 healthy women at their first and second half of pregnancy; the third group includes 60 women with pregnancy developing against the background of noncomplicated insulin-dependent diabetus mellitus during their first and second half of pregnancy. In the healthy women there are not any significant changes in most of the parameters of the microcirculatory bed during the first half of their pregnancy, and in the second half of their pregnancy a great density of the blood vascular bed is determined. Diameters of all vessels in the hemomicrocirculatory bed are significantly increased, as compared to those in the control group. In the women suffering from diabetus mellitus, during the first half of pregnancy certain changes in the picture of the vascular network is observed, as well as an increased convolution. uneveness in distribution of blood vessels; in the second half of their pregnancy a pronounced deformity of the vacular network is observed, decreasing diameter of afferent vessels and an essential dilatation of postcapillaries and venules are registered. Certain signs of aggregation of blood formed elements is noted.     

Morphology of central compartment and vasculature of the gills of Lepidosiren paradoxa (Fitzinger)

The four paired gill arches of the South American lungfish Lepidosiren paradoxa contain single branchial arteries directly connecting dorsal and ventral arteries. In gill arches 3 and 4 the branchial arteries also supply looped arlerioles and capillaries to much-reduced gill filaments. Regulation of blood between these routes is thought to be by alteration of vascular resistance. Within the filaments, extensive subepithelial capillary networks and numerous small pumps connect lymphatic vessels in the central connective tissue compartment with venules which, in turn, drain to paired branchial veins.
The features of the endothelium of many of the filament blood vessels suggest extensive transporting, haematolytic and granulopoeitic functions. Large numbers of macrophages pack the connective tissue. Many contain extensive quantities of haemosiderin.     

Mathematical analysis of several parameters of the microcirculatory bed of muscles and muscular organs]

The authors propose a complex mathematical method for analysis of the microcirculatory link of the vascular bed of muscles and muscular organs. On the basis of the data of the amount and diameters of the vessels the method permits calculating the following parameters according to the proposed pattern of the table-report and formulas: 1. the square surface of the cross-section of the vascular bed; 2. the square surface of the exchange or contact with the tissue surface of the vascular bed; 3. the density of the vascular network in percentage; 4. the blood volume in the vascular bed in volumetric units and average data; 5. the blood volume in one vessel on the average; 6. the volume of the tissue fed by one vessel; 7. the volume of blood per a unit of the surface of the vascular wall.     

Microcirculation-on-a-Chip: A Microfluidic Platform for Assaying Blood- and Lymphatic-Vessel Permeability

We developed a microfluidic model of microcirculation containing both blood and lymphatic vessels for examining vascular permeability. The designed microfluidic device harbors upper and lower channels that are partly aligned and are separated by a porous membrane, and on this membrane, blood vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs) were cocultured back-to-back. At cell-cell junctions of both BECs and LECs, claudin-5 and VE-cadherin were detected. The permeability coefficient measured here was lower than the value reported for isolated mammalian venules. Moreover, our results showed that the flow culture established in the device promoted the formation of endothelial cell-cell junctions, and that treatment with histamine, an inflammation-promoting substance, induced changes in the localization of tight and adherens junction-associated proteins and an increase in vascular permeability in the microdevice. These findings indicated that both BECs and LECs appeared to retain their functions in the microfluidic coculture platform. Using this microcirculation device, the vascular damage induced by habu snake venom was successfully assayed, and the assay time was reduced from 24 h to 30 min. This is the first report of a microcirculation model in which BECs and LECs were cocultured. Because the micromodel includes lymphatic vessels in addition to blood vessels, the model can be used to evaluate both vascular permeability and lymphatic return rate.     

Three-Dimensional Imaging of Prox1-EGFP Transgenic Mouse Gonads Reveals Divergent Modes of Lymphangiogenesis in the Testis and Ovary

The lymphatic vasculature forms a specialized part of the circulatory system, being essential for maintaining tissue fluid homeostasis and for transport of hormones, macromolecules, and immune cells. Although lymphatic vessels are assumed to play an important role in most tissues, their morphogenesis and function in the gonads remains poorly understood. Here we have exploited a lymphatic-specific Prox1-EGFP reporter mouse model and optical projection tomography technology to characterize both the temporal and spatial development of the lymphatic vessel network in mouse testes and ovaries. We find that lymphangiogenesis in the testis is initiated during late gestation, but in contrast to other organs, lymphatic vessels remain confined to the testis cap and, unlike blood vessels, do not infiltrate the entire organ. Conversely, lymphatic vessels invade the ovarian tissue, beginning postnatally, and sprouting from preexisting lymphatic vessels at the extraovarian rete. The ovary develops a rich network of lymphatic vessels, extending from the medulla into the surrounding cortex adjacent to developing follicles. This study reveals distinct patterns of lymphangiogenesis in the testes and ovaries and will serve as the basis for the identification of the divergent molecular pathways that control morphogenesis and the function of the lymphatic vasculature in these two organs.     

Ontogeny of human fetal lymph nodes.

Developing lymph nodes from 30 human embryos and fetuses with crown-rump lengths (CRL) of 18 mm (5.6 wk) to 245 mm (26 wk) were examined by light microscopy. The nodes were embedded in araldite, and the sections examined were approximately 1 mu in thickness. The development of nodes was divided into three stages: 1. the lymphatic plexus and connective tissue invagination (30 mm to 67 mm CRL); 2. the early fetal lymph node (43 mm to ,5 mm CRL); and 3. the late fetal lymph node (CRL greater than 75 mm). The lymphatic plexus was formed by connective tissue invaginations and bridges which divided a lymph sac into a meshwork of channels and spaces. Connective tissue invaginations were endothelially-lined and were surrounded by lymphatic space. Reticular cells, macrophages, and blood vessels were found in these invaginations. Early fetal lymph nodes were formed from invaginations when the cellular density and lymphocyte content increased. The lymphatic space surrounding the early node was the developing subcapsular sinus. With further development the early node became packed with lymphocytes, increasing the cellular density and size of the node. The connective tissue surrounding the subcapsular sinus condensed to form the capsule. Afferent lymphatic vessels pierced the capsule. Capillaries, veins, postcapillary venules, and occasional arteries were found in early and late nodes.     

Genesis and pathogenesis of lymphatic vessels

The lymphatic system is generally regarded as supplementary to the blood vascular system, in that it transports interstitial fluid, macromolecules, and immune cells back into the blood. However, in insects, the open hemolymphatic (or lymphohematic) system ensures the circulation of immune cells and interstitial fluid through the body. The Drosophila homolog of the mammalian vascular endothelial growth factor receptor (VEGFR) gene family is expressed in hemocytes, suggesting a close relationship to the endothelium that develops later in phylogeny. Lymph hearts are typical organs for the propulsion of lymph in lower vertebrates and are still transiently present in birds. The lymphatic endothelial marker VEGFR-3 is transiently expressed in embryonic blood vessels and is crucial for their development. We therefore regard the question of whether the blood vascular system or the lymphatic system is primary or secondary as open. Future molecular comparisons should be performed without any bias based on the current prevalence of the blood vascular system over the lymphatic system. Here, we give an overview of the structure, function, and development of the lymphatics, with special emphasis on the recently discovered lymphangiogenic growth factors.     

Fine structure of venules and lymphatics of the dog thyroid gland]

The endothelial cells of the venules (collecting venules) of the thyroid gland of the dog are very thin and show fenestrations. In their wall, however, more 'primitive' smooth muscle cell layers are recognizable. The endothelial cells in the lymphatic vessels are also thin, but continuous. They have all known morphological characteristics of these vessels. The fenestrae of the venules' endothelium could facilitate hormone passage from the interstitial tissue into the blood stream.     

Ang-Tie轴在血管和淋巴系统相关疾病中作用的研究进展

形成血管和淋巴管内层的内皮细胞是脉管系统的重要组成部分,并参与血管和淋巴系统疾病的发病机制。内皮细胞上的血管生成素(Angiopoietin,Ang)-具有免疫球蛋白和表皮生长因子同源性结构域的酪氨酸蛋白激酶(Tyrosine kinase receptors with immunoglobulin and EGF homology domains,Tie)轴是除了血管内皮生长因子受体途径外胚胎心血管和淋巴发育所必需的第二种内皮细胞特异性配体-受体信号传导系统。Ang-Tie轴参与调节产后血管生成与重塑、血管通透性和炎症,以维持血管平衡,因此,该系统在许多血管和淋巴系统疾病中发挥重要的作用。针对近年来Ang-Tie轴在血管和淋巴系统相关疾病中作用的研究进展,文中系统论述了Ang-Tie轴在炎症诱导的血管通透性、血管重塑、眼部新生脉管、剪切应力反应、动脉粥样硬化和肿瘤血管生成和转移中的作用,并总结了涉及Ang-Tie轴的相关治疗性抗体、重组蛋白和小分子药物。     

Regulation of lymphangiogenesis--from cell fate determination to vessel remodeling

Lymphatic vessels are important for the maintenance of normal tissue fluid balance, immune surveillance and adsorption of digested fats. During the past decade, the identification of lymphatic-specific markers and growth factors has enabled detailed studies of the lymphatic system, and gain- and loss-of-function experiments have greatly increased our understanding of the mechanisms of normal lymphatic development. Understanding the basic biology has provided novel insights into the pathologic conditions of the lymphatic system that contribute to lymphedema, inflammation or lymphatic metastasis, and opened possibilities for the development of better therapeutic strategies. Here we review the current knowledge about the molecular mechanisms regulating the development of the lymphatic vasculature; of the differentiation of lymphatic endothelial cells, of the regulation of the growth of lymphatic vessels, and of remodeling of the vasculature into a network consisting of lymphatic capillaries and collecting lymphatic vessels. Furthermore, we will discuss the molecular mechanisms involved in the pathological conditions of the lymphatic vessels.     

Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.     

Intravital two-photon microscopy of lymphatic vessel development and function using a transgenic Prox1 promoter-directed mOrange2 reporter mouse

Lymphatic vessels, the second vascular system of higher vertebrates, are indispensable for fluid tissue homoeostasis, dietary fat resorption and immune surveillance. Not only are lymphatic vessels formed during fetal development, when the lymphatic endothelium differentiates and separates from blood endothelial cells, but also lymphangiogenesis occurs during adult life under conditions of inflammation, wound healing and tumour formation. Under all of these conditions, haemopoietic cells can exert instructive influences on lymph vessel growth and are essential for the vital separation of blood and lymphatic vessels. LECs (lymphatic endothelial cells) are characterized by expression of a number of unique genes that distinguish them from blood endothelium and can be utilized to drive reporter genes in a lymph endothelial-specific fashion. In the present paper, we describe the Prox1 (prospero homeobox protein 1) promoter-driven expression of the fluorescent protein mOrange2, which allows the specific intravital visualization of lymph vessel growth and behaviour during mouse fetal development and in adult mice.     

Transgenic expression of Angiopoietin 1 in the liver leads to changes in lymphatic and blood vessel architecture

To investigate the possible role of the Angiopoietins in vessel remodelling, we overexpressed one of the angiopoietins, Angiopoietin-1 (Ang1), in the hepatocytes of mice by means of the conditional binary transgenic system. Animals were examined by Doppler ultrasound, and dissected livers were analyzed by immunohistochemical staining. Double transgenic mice presented with enlarged spleens and kidneys, enlarged, disorganized blood vessels located near the surface of the liver, sprouting, dilation, and disorganization of liver lymphatics, and turbulent flow in about 1/4 of the blood vessels sampled. Most of these characteristics completely resolved within 12 weeks of turning off the expression of the Ang1 transgene, illustrating a dependence on the continual presence of Ang1 for maintenance of the vascular phenotype. Conditional Angiopoietin-1 overexpression in the liver of mice leads to a phenotype highly reminiscent of portal hypertension illustrating that Ang1 can drive both vascular and lymphatic vessel remodelling and may play a role in portal hypertension.     

Distribution of lymphatic vessels in mouse thymus: immunofluorescence analysis

Thymic blood and lymphatic vessels in humans and laboratory animals have been investigated in morphological studies. However, occasionally a clear distinction between blood vessels and lymphatic vessels cannot be made from morphological characteristics of the vasculature. To visualize thymic lymphatics in normal adult BALB/c mice, we used antibodies against specific markers of lymphatic endothelial cells. Expression of vascular endothelial growth factor receptor–3 (VEGFR–3) was detected throughout the thymus, i.e., the capsule, cortex, and medulla. Most thymic lymphatics were present in capillaries of ~20 μm in caliber. The plexuses of lymphatic capillaries were occasionally detectable. Lymphatic vessels were frequently adjacent to CD31–positive blood vessels, and some lymphatic vessels were seen in the immediate vicinity of or within the perivascular spaces around postcapillary venules. The identity of VEGFR–3–positive vessels as lymphatics was further confirmed by staining with additional markers: LYVE–1, Prox–1, neuropilin–2, and secondary lymphoid tissue chemokine (SLC). The distributions of LYVE–1 were similar to those of VEGFR–3. Most lymphatic vessels were also identified by Prox–1. Neuropilin–2 was restricted to lymphatic vessels in the thymus. The most abundant expression of SLC in the thymus was in medullar epithelial cells; SLC was also expressed in lymphatic vessels and blood vessels. Thus, lymphatic endothelium in mouse thymus was characterized by positive staining with antibodies to VEGFR–3, LYVE–1, Prox–1, neuropilin–2, or SLC, but not with an antibody to CD31. Our results suggest the presence of lymphatic capillary networks throughout the thymus.     

Local adjustment of blood and lymph circulation in the hormonal regulation of reproduction in female pigs--facts, conclusions and suggestions for future research

Arteries, veins, capillaries and lymphatic vessels situated in the mesovarium and mesosalpinx of domestic animal species (pig, cow, sheep) form the periovarian vascular complex. Particular components of the periovarian vascular complex interact functionally and morphologically creating a specific environment for numerous physiological processes. The complex plays an essential role in the system of the retrograde transfer of the ovarian hormones. This phenomenon is especially well documented in pigs. The efficiency of the retrograde transfer of estradiol and progesterone from blood and lymph leaving the gonad to blood of the ovarian artery (expressed as percentage of their concentration in the ovarian venous blood) as well as the rate of the retrograde transfer of these hormones to the ovary (measured in nanograms or picograms per minute) is presented and discussed in this paper. No simple relationship was found between hormone concentration in ovarian venous effluent and the efficiency or the rate of their retrograde transfer during the estrous cycle. It appears that two processes contribute to the highly efficient retrograde transfer of ovarian hormones into the ovary in the periovarian vascular complex: 1/ direct hormone permeation from the ovarian vein into the adjacent branches of the ovarian artery through the counter-current mechanism; 2/ indirect permeation of ovarian hormones consisting of two stages. The first stage includes the permeation of hormones from lymph leaving the ovary via the subovarian lymphatic vascular network as well as lymph and venous blood, leaving the mesosalpinx and going to capillaries and tiny venous vessels in the entire mesovarium. These tiny mesovarium vessels connect and then branch out again to form the veno-venous network on the surface of branches of the ovarian artery. The second stage includes the permeation of hormones from the veno-venous blood into the branches of the ovarian artery. The authors present a hypothesis that the retrograde transfer of ovarian hormones may participate in the feedback regulation of ovarian function. The relationship between the retrograde transfer of ovarian hormones in the area of periovarian vascular complex and local elevation of steroid hormone concentrations in blood supplying the oviduct and uterus is presented. The paper also includes suggestions for future research.     

A coupled discrete/continuum model for describing cancer-therapeutic transport in the lung

We propose a computational simulation framework for describing cancer-therapeutic transport in the lung. A discrete vascular graph model (VGM) is coupled to a double-continuum model (DCM) to determine the amount of administered therapeutic agent that will reach the cancer cells. An alveolar cell carcinoma is considered. The processes in the bigger blood vessels (arteries, arterioles, venules and veins) are described by the VGM. The processes in the alveolar capillaries and the surrounding tissue are represented by a continuum approach for porous media. The system of equations of the coupled discrete/continuum model contains terms that account for degradation processes of the therapeutic agent, the reduction of the number of drug molecules by the lymphatic system and the interaction of the drug with the tissue cells. The functionality of the coupled discrete/continuum model is demonstrated in example simulations using simplified pulmonary vascular networks, which are designed to show-off the capabilities of the model rather than being physiologically accurate.