Rab35 Governs Apicobasal Polarity Through Regulation of Actin Dynamics During Sprouting Angiogenesis

In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35s primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization, which is required to setup proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent regulator of actin architecture during blood vessel development.

Mathematical simulation and prediction of tumor volume using RBF artificial neural network at different circumstances in the tumor microenvironment

Uncontrolled proliferation of cells in a tissue caused by genetic mutations inside a cell is referred to as a tumor. A tumor which grows rapidly encounters a barrier when it grows to a certain size in presence of preexisting vasculature. This is the time when it has to find a way to go on the growth. The tumor starts to secrete tumor angiogenic factors (TAFs) and stimulate preexisting vessels to grow new sprouts. These new sprouts will find their way to the tumor in the extracellular matrix (ECM) by the gradient of TAF. As these new capillaries anastomose and reach tumor, fresh oxygen is available for the tumor and it will reinitiate the growth. Number of initial sprouts, distance of initial tumor cells from the vessel(s) and initial density of the tumor at the time of sprout formation are questions which are to be investigated. In the present study, the aim is to find the response of tumor cells and vessels to the reciprocal effects of each other in different circumstances in the tissue. Together with a mathematical formulation, a radial basis function (RBF) neural network is established to predict the number of tumor cells at different circumstances including size and distance of initial tumors from the parent vessel. A final formulation is given for the final number of tumor cells as a function of initial tumor size and distance between a parent vessel and a tumor. Results of this simulation demonstrate that, increasing the distance between a tumor and a parent vessel decreases the number of final tumor cells. Specially, this decrement becomes faster beyond a certain distance. Moreover, initial tumors in bigger domains must become much bigger before inducing angiogenesis which makes it harder for them to survive.

3D printing of self-standing and vascular supportive multi-material hydrogel structures for organ engineering

Three dimensional printable formulation of self-standing and vascular-supportive structures using multi-materials suitable for organ engineering is of great importance and highly challengeable, but, it could advance the 3D printing scenario from printable shape to functional unit of human body. In this study, the authors report a 3D printable formulation of such self-standing and vascular-supportive structures using an in-house formulated multi-material combination of albumen/alginate/gelatin (A-SA-Gel)-based hydrogel. The rheological properties and relaxation behavior of hydrogels were analyzed prior to the printing process. The suitability of the hydrogel in 3D printing of various customizable and self-standing structures, including a human ear model, was examined by extrusion-based 3D printing. The structural, mechanical, and physicochemical properties of the printed scaffolds were studied systematically. Results supported the 3D printability of the formulated hydrogel with self-standing structures, which are customizable to a specific need. In vitro cell experiment showed that the formulated hydrogel has excellent biocompatibility and vascular supportive behavior with the extent of endothelial sprout formation when tested with human umbilical vein endothelial cells. In conclusion, the present study demonstrated the suitability of the extrusion-based 3D printing technique for manufacturing complex shapes and structures using multi-materials with high fidelity, which have great potential in organ engineering.

The Role of Heparan Sulfate and Neuropilin 2 in VEGFA Signaling in Human Endothelial Tip Cells and Non-Tip Cells during Angiogenesis In Vitro

During angiogenesis, vascular endothelial growth factor A (VEGFA) regulates endothelial cell (EC) survival, tip cell formation, and stalk cell proliferation via VEGF receptor 2 (VEGFR2). VEGFR2 can interact with VEGFR2 co-receptors such as heparan sulfate proteoglycans (HSPGs) and neuropilin 2 (NRP2), but the exact roles of these co-receptors, or of sulfatase 2 (SULF2), an enzyme that removes sulfate groups from HSPGs and inhibits HSPG-mediated uptake of very low density lipoprotein (VLDL), in angiogenesis and tip cell biology are unknown. In the present study, we investigated whether the modulation of binding of VEGFA to VEGFR2 by knockdown of SULF2 or NRP2 affects sprouting angiogenesis, tip cell formation, proliferation of non-tip cells, and EC survival, or uptake of VLDL. To this end, we employed VEGFA splice variant 121, which lacks an HSPG binding domain, and VEGFA splice variant 165, which does have this domain, in in vitro models of angiogenic tip cells and vascular sprouting. We conclude that VEGFA165 and VEGFA121 have similar inducing effects on tip cells and sprouting in vitro, and that the binding of VEGFA165 to HSPGs in the extracellular matrix does not seem to play a role, as knockdown of SULF2 did not alter these effects. Co-binding of NRP2 appears to regulate VEGFA–VEGFR2-induced sprout initiation, but not tip cell formation. Finally, as the addition of VLDL increased sprout formation but not tip cell formation, and as VLDL uptake was limited to non-tip cells, our findings suggest that VLDL plays a role in sprout formation by providing biomass for stalk cell proliferation.

The adaptor protein Grb2b is an essential modulator for lympho-venous sprout formation in the zebrafish trunk

Vegfc/Vegfr3 signaling is critical for lymphangiogenesis, the sprouting of lymphatic vessels. In zebrafish, cells sprouting from the posterior cardinal vein can either form lymphatic precursor cells or contribute to intersegmental vein formation. Both, the Vegfc-dependent differential induction of Prox1a in sprouting cells as well as a Notch-mediated pre-pattern within intersegmental vessels have been associated with the regulation of secondary sprout behavior. However, how exactly a differential lymphatic versus venous sprout cell behavior is achieved is not fully understood. Here, we characterize a zebrafish mutant in the adaptor protein Grb2b, and demonstrate through genetic interaction studies that Grb2b acts within the Vegfr3 pathway. Mutant embryos exhibit phenotypes that are consistent with reduced Vegfr3 signaling outputs prior to the sprouting of endothelial cells from the vein. During secondary sprouting stages, loss of grb2b leads to defective cell behaviors resulting in a loss of parachordal lymphangioblasts, while only partially affecting the number of intersegmental veins. A second GRB2 zebrafish ortholog, grb2a, contributes to the development of lymphatic structures in the meninges and in the head, but not in the trunk. Our results illustrate an essential role of Grb2b in vivo for cell migration to the horizontal myoseptum and for the correct formation of the lymphatic vasculature, while being less critically required in intersegmental vein formation. Thus, there appear to be higher requirements for Grb2b and therefore Vegfr3 downstream signaling levels in lymphatic versus vein precursor-generating sprouts.

Varieties of sweet potato and features of their cultivation technology

В 2017–2019 годах изучены сортообразцы сладкого картофеля на староорошаемых лугово-сероземных почвах Зарафшанской долины. Цель исследований – комплексная оценка сортообразцов батата в условиях Зарафшанской долины по скороспелости, росту, развитию, интенсивному размножению, формированию урожая, компактности клубней в гнезде, продуктивности, урожайности и лежкости клубней и выделение из них перспективных, а также разработка приемов агротехнологии получения высоких урожаев для данных условий. Почвы опытного участка характеризуются благоприятными агрофизическими и водными свойствами и низким содержанием гумуса, нитратного азота, подвижного фосфора и содержанием обменного калия от низкого до среднего. Сравнивали 18 сортообразцов батата. Выделенные сортообразцы изучали при ширине междурядий 70 и 90 см со схемой 70×25 и 90×20 см по 1, 2 и 3 растения в гнезде. Для получения рассады сортообразцов батата брали по 40 клубней и высаживали 20–22 февраля в пленочной теплице при температуре 15–18 °C, заделывая их на глубину 3–5 см. Влажность почвы поддерживали на уровне 65–70%. Через 7–10 дней после высадки почки начинают прорастать, а через 43–48 дней формируется рассада высотой 12–15 см, готовая для высадки в поле. Высадка рассады, как и других рассадных культур (томата, перца, баклажана) – по схеме 70×20–25 см во второй-третьей декаде апреля. Уход включал в себя междурядную обработку (культивация), прополку, борьбу с сорняками, подкормку, поливы. Уборка урожая – в конце сентября-начале октября с помощью картофелекопателя. В результате исследований выделились сорта по скороспелости, дружности формирования ростков, ускоренному размножению, продуктивности, компактности клубней в гнезде и с высоким товарным урожаем – Сочакинур, Хар-Бей и Япон. Возделывание этих сортообразцов по схеме 70×25 и 90×20 см способствует получению товарного урожая высокого качества не менее 43–48 т/га. In 2017–2019, studies were conducted on the study of varieties of sweet potatoes on old-irrigated meadow-gray-earth soils of the Zarafshan valley. The purpose of the research is a comprehensive assessment of sweet potato varieties in the conditions of the Zarafshan valley in terms of early maturity, growth, development, intensive reproduction, yield formation, compactness of tubers in the nest, productivity, yield and keeping quality of tubers and the selection of promising ones, as well as the development of methods of agricultural technology for obtaining high yields for given conditions. The soil is characterized by favorable agrophysical and water properties and low content of humus, nitrate nitrogen, mobile phosphorus and a low-average content of exchange potassium. Eighteen sweet potato varieties were compared. The selected variety samples were studied with a row spacing of 70 and 90 cm with a scheme of 70×25 and 90×20 cm for 1, 2 and 3 seedlings in the nest. To obtain seedlings of sweet potato varieties, 40 tubers were taken and planted on February 20–22 in a film greenhouse at a temperature of 15–18 °C, seeding to a depth of 3–5 cm, the soil moisture was maintained at 65–70%. 7–10 days after planting, the buds began to germinate, and after 43–48 days, seedlings were formed with a height of 12–15 cm, which is ready for planting in the field. Planting seedlings in the field is carried out, as well as other seedlings (tomato, pepper, eggplant) according to the scheme 70x20–25 cm in the second or third decade of April. Growing includes inter-row processing (cultivation), weeding, weed control, top dressing, watering. Harvesting in late September, early October with a potato digger. As a result of the research, varieties were identified in terms of early maturity, sprout formation, accelerated reproduction, productivity, compactness of tubers in the nest and ensuring a high marketable yield – Sochakinur, Xar-Bey, and Japon. The cultivation of these varieties according to the scheme 70×25 and 90×20 cm contributes to the production of a commodity crop at 43–48 t/ha with good qualities.

The β1-integrin plays a key role in LEC invasion in an optimized 3-D collagen matrix model

Lymphangiogenesis, or formation of new lymphatic vessels, is a tightly regulated process that is controlled by growth factor signaling and biomechanical cues. Lymphatic endothelial cells (LECs) undergo remodeling, migration, and proliferation to invade the surrounding extracellular matrix (ECM) during both physiological and pathological lymphangiogenesis. This study optimized conditions for an in vitro three-dimensional (3-D) collagen-based model that induced LEC invasion and recapitulated physiological formation of lymphatic capillaries with lumens. Invasion of LECs was enhanced in the presence of sphingosine 1-phosphate (S1P). Effects of various known lymphangiogenic factors, vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), interleukin (IL)-8, and hepatocyte growth factor (HGF), were tested on LEC sprout formation synergistically with VEGF-C. Several of these growth factors significantly enhanced LEC invasion, and synergistic effects of some of these further enhanced the sprouting density and lumen volume. To determine the contribution of specific ECM components, we analyzed the expression of different integrin subunits. Basal expressions of the integrin α5- and integrin β1-subunits were high in LECs. The addition of fibronectin, which mediates cellular responses through these integrins, enhanced LEC sprouting density and sprout length dose-dependently. siRNA-mediated knockdown of the integrin β1-subunit suppressed LEC invasion and also inhibited VEGF receptor (VEGFR)3 and ERK activation. Furthermore, exposing LECs to the inflammatory mediator lipopolysaccharide (LPS) inhibited sprouting. This optimized model for LEC invasion includes S1P, VEGF-C, and fibronectin within a 3-D collagen matrix, along with VEGF-C, VEGF-A, bFGF, and HGF in the culture medium, and provides a useful tool to investigate the functional effect of various lymphangiogenic factors and inhibitors.

ErMiao San Inhibits Angiogenesis in Rheumatoid Arthritis by Suppressing JAK/STAT Signaling Pathways

ErMiao San (EMS) is composed of the Cortex Phellodendri chinensis and Atractylodes lancea, and it has the function of eliminating heat and excreting dampness in terms of traditional Chinese medicine to damp heat syndrome. Previous reports indicate that EMS possesses anti-inflammatory activity; however, its action on angiogenesis of rheumatoid arthritis (RA) has not been clarified. The present study aims to determine the antiangiogenic activity of EMS in collagen-induced arthritis (CIA) mice and in various angiogenesis models. Our data showed that EMS (5 g/kg) markedly reduced the immature blood vessels in synovial membrane tissues of inflamed joints from CIA mice. It also inhibited vascular endothelial growth factor (VEGF)-induced microvessel sprout formation ex vivo. Meanwhile, EMS suppressed VEGF-induced migration, invasion, adhesion, and tube formation of human umbilical vein endothelial cells (HUVECs). Moreover, EMS significantly reduced the expression of angiogenic activators including interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) in synovium of CIA mice. More interestingly, EMS blocked the autophosphorylation of VEGF-induced JAK1, STAT1, and STAT6 in CIA mice and VEGF-induced HUVECs. These findings suggest for the first time that EMS possesses the antiangiogenic effect in RA in vivo, ex vivo, and in vitro by interrupting the targeting of JAK/STAT activation.

VE-Cadherin and Anastomosis of Blood Vessels Formed by Dental Stem Cells

It is known that dental pulp stem cells (DPSCs) can be induced to differentiate into vasculogenic endothelial (VE) cells. However, the process that results in sprouting and anastomosis of DPSC-derived vessels remains unclear. Here, we performed studies to understand the mechanisms underpinning the anastomosis of the host vasculature with blood vessels generated by DPSCs (a model for mesenchymal stem cells). VE-cadherin–silenced primary human DPSCs seeded in tooth slice/scaffolds and transplanted into the subcutaneous space of immunodeficient mice generated fewer functional blood vessels (i.e., anastomosed with the host vasculature) than control DPSCs transduced with scrambled sequences. Both VE-cadherin–silenced and mitogen-activated protein kinase kinase 1 (MEK1)–silenced cells showed a decrease in the number of capillary sprouts in vitro. Interestingly, DPSC stably transduced with a VE-cadherin reporter demonstrated that vascular endothelial growth factor (VEGF) induces VE-cadherin expression in sprouting DPSCs undergoing anastomosis, but not in quiescent DPSCs. To begin to understand the mechanisms regulating VE-cadherin, we stably silenced MEK1 and observed that VEGF was no longer able to induce VE-cadherin expression and capillary sprout formation. Notably ERG, a transcriptional factor downstream from MEK/ERK, binds to the promoter region of VE-cadherin (chip assay) and is induced by VEGF in DPSCs. Collectively, these data defined a signaling pathway triggered by VEGF that results in phosphorylation of MEK1/ERK and activation of ERG leading to expression of VE-cadherin, which is required for anastomosis of DPSC-derived blood vessels. In conclusion, these results unveiled a signaling pathway that enables the generation of functional blood vessels upon vasculogenic differentiation of DPSCs.

Integrated assessment of introduction prospects of Deutzia Thunb. genus representatives in the South-Ural Botanical Garden-Institute (Ufa)

The paper contains results of introduced study of species, hybrids and sorts of Deutzia Thunb. genus in the South-Ural botanical garden-institute (Ufa). The collection of Deutzia is one of the largest among ornamental shrubs in the botanical garden. The objects of researches were 12 species, 10 hybrids and sorts of Deutzia . Introduced researches of all taxons were conducted within 10 years (2007-2017). Throughout the entire period of observations the following indicators were annually estimated: lignification of escapes, winter hardiness, preservation of a form of growth, sprout-formation ability, a gain in height, generative development, possible ways of reproduction in the culture. The analysis of the obtained data shows that specific taxons are belong to three groups: quite perspective, perspective and less perspective. The greatest possible indicators characterize 3 species - D. amurensis , D. glabrata, D. parviflora . All of them can be recommended for broad application in gardening of settlements of the Bashkortostan Republic. Perspective and less perspective groups contained 4 species each. Hybrids and sorts in the collection belong to three groups: perspective (II), less perspective (III) completely unusable (VI); in the latter group contains one hybrid.