Targeting FoxO1 in traditional Chinese medicine for the treatment of diabetes.

Traditional Chinese medicine (TCM) encompasses treatment strategies for diabetes, which is referred to as "Consumptive Thirsty" syndrome. Recently, there has been discovery regarding the mapping between TCM and signaling molecules, which has revealed a remarkable consistency between TCM and modern medicine from a molecular perspective. In this manuscript, we have summarized the etiology and treatment strategies for diabetes in TCM and have examined these strategies in the context of molecular mechanisms. Our review demonstrates that the targeting molecule of TCM for the treatment of diabetes is FoxO1, a transcription factor that plays a pivotal role in regulating gluconeogenesis and glycogenolysis. TCM ranks the development of diabetes into three stages and utilizes different herbal formulas to control FoxO1 accordingly. At Stage 1, TCM inhibits FoxO1 by lowering its expression in the lung. At Stage 2, TCM increases the expression of FoxO1 by suppressing its activity in the stomach. At Stage 3, TCM utilizes the famous herbal formula Liuwei Dihuang Pill to amplify the expression of FoxO1, and to enhance the concentrations of potassium, phosphorus, and Wnt, but to reduce the concentration of calcium. These TCM treatment strategies are in accordance with corresponding mechanisms in modern medicine.

Zinc regulates vascular endothelial cell activity through zinc-sensing receptor ZnR/GPR39.

Zn2+ is an essential element for cell survival/growth, and its deficiency is linked to many disorders. Extracellular Zn2+ concentration changes participate in modulating fundamental cellular processes such as proliferation, secretion, ion transport, and cell signal transduction in a mechanism that is not well understood. Here, we hypothesize that the Zn2+-sensing receptor ZnR/G protein-coupled receptor 39 (GPR39), found in tissues where dynamic Zn2+ homeostasis takes place, enables extracellular Zn2+ to trigger intracellular signaling pathways regulating key cell functions in vascular cells. Thus, we investigated how extracellular Zn2+ regulates cell viability, proliferation, motility, angiogenesis, vascular tone, and inflammation through ZnR/GPR39 in endothelial cells. Knockdown of GPR39 through siRNA largely abolished Zn2+-triggered cellular activity changes, Ca2+ responses, as well as the downstream activation of Gαq-PLC pathways. Extracellular Zn2+ promoted vascular cell survival/growth through activation of cAMP and Akt as well as overexpressing of platelet-derived growth factor-α receptor and vascular endothelial growth factor A. It also enhanced cell adhesion and mobility, endothelial tubule formation, and cytoskeletal reorganization. Such effects from extracellular Zn2+ were not observed in GPR39-/- endothelial cells. Zn2+ also regulated inflammation-related key molecules such as heme oxygenase-1, selectin L, IL-10, and platelet endothelial cell adhesion molecule 1, as well as vascular tone-related prostaglandin I2 synthase and nitric oxide synthase-3. In sum, extracellular Zn2+ regulates endothelial cell activity in a ZnR/GPR39-dependent manner and through the downstream Gαq-PLC pathways. Thus, ZnR/GPR39 may be a therapeutic target for regulating endothelial activity.

Endothelial glycocalyx-the battleground for complications of sepsis and kidney injury.

After briefly discussing endothelial glycocalyx and its role in vascular physiology and renal disease, this overview focuses on its degradation very early in the course of microbial sepsis. We describe our recently proposed mechanism for glycocalyx degradation induced by exocytosis of lysosome-related organelles and release of their cargo. Notably, an intermediate in nitric oxide synthesis, NG-hydroxy-l-arginine, shows efficacy in curtailing exocytosis of these organelles and improvement in animal survival. These data not only depict a novel mechanism responsible for very early glycocalyx degradation, but may also outline a potential preventive therapy. The second issue discussed in this article is related to the therapeutic acceleration of restoration of already degraded endothelial glycocalyx. Here, using as an example our recent findings obtained with sulodexide, we illustrate the importance of the expedited repair of degraded endothelial glycocalyx for the survival of animals with severe sepsis. These two focal points of the review on glycocalyx may not only have broader disease applicability, but they may also provide additional evidence to buttress the idea of the importance of endothelial glycocalyx and its maintenance and repair in the prevention and treatment of an array of renal and nonrenal diseases.

Tumor Metastasis in the Microcirculation.

Tumor cell metastasis through blood circulation is a complex process and is one of the great challenges in cancer research as metastatic spread is responsible for ∼90% of cancer-related mortality. Tumor cell intravasation into, arrest and adhesion at, and extravasation from the microvessel walls are critical steps in metastatic spread. Understanding these steps may lead to new therapeutic concepts for tumor metastasis. Vascular endothelium forming the microvessel wall and the glycocalyx layer at its surface are the principal barriers to and regulators of the material exchange between circulating blood and body tissues. The cleft between adjacent endothelial cells is the principal pathway for water and solute transport through the microvessel wall in health. Recently, this cleft has been found to be the location for tumor cell adhesion and extravasation. The blood-flow-induced hydrodynamic factors such as shear rates and stresses, shear rate and stress gradients, as well as vorticities, especially at the branches and turns of microvasculatures, also play important roles in tumor cell arrest and adhesion. This chapter therefore reports the current advances from in vivo animal studies and in vitro culture cell studies to demonstrate how the endothelial integrity or microvascular permeability, hydrodynamic factors, microvascular geometry, cell adhesion molecules, and surrounding extracellular matrix affect critical steps of tumor metastasis in the microcirculation.

Transport Across the Blood-Brain Barrier.

The blood-brain barrier (BBB) is a dynamic barrier essential for maintaining the microenvironment of the brain. Although the special anatomical features of the BBB determine its protective role for the central nervous system (CNS) from blood-borne neurotoxins, however, the BBB extremely limits the therapeutic efficacy of drugs into the CNS, which greatly hinders the treatment of major brain diseases. This chapter summarized the unique structures of the BBB; described a variety of in vivo and in vitro experimental methods for determining the transport properties of the BBB and the permeability of the BBB to water, ions, and solutes including nutrients, therapeutic agents, and drug carriers; and presented recently developed mathematical models which quantitatively correlate the anatomical structures of the BBB with its barrier functions. Recent findings for modulation of the BBB permeability by chemical and physical stimuli were described. Finally, drug delivery strategies through specific trans-BBB routes were discussed.

The Role of Endothelial Surface Glycocalyx in Mechanosensing and Transduction.

The endothelial cells (ECs) forming the inner wall of every blood vessel are constantly exposed to the mechanical forces generated by blood flow. The EC responses to these hemodynamic forces play a critical role in the homeostasis of the circulatory system. A variety of mechanosensors and transducers, locating on the EC surface, intra- and trans-EC membrane, and within the EC cytoskeleton, have thus been identified to ensure proper functions of ECs. Among them, the most recent candidate is the endothelial surface glycocalyx (ESG), which is a matrix-like thin layer covering the luminal surface of the EC. It consists of various proteoglycans, glycosaminoglycans, and plasma proteins and is close to other prominent EC mechanosensors and transducers. This chapter summarizes the ESG composition, thickness, and structure observed by different labeling and visualization techniques and in different types of vessels. It also presents the literature in determining the ESG mechanical properties by atomic force microscopy and optical tweezers. The molecular mechanisms by which the ESG plays the role in EC mechanosensing and transduction are described as well as the ESG remodeling by shear stress, the actin cytoskeleton, the membrane rafts, the angiogenic factors, and the sphingosine-1-phosphate.

Blood-Brain Barrier Integrity and Clearance of Amyloid-ß from the BBB.

Alzheimer's disease, a type of dementia, affects memory, behavior, and cognitive processes in affected individuals. It is one of the prominent diseases, accounting for 60-80% of dementia cases and affecting a significant population of persons over the age of 65 years. While rare, Alzheimer's disease (AD) may affect the younger population as well. With such a widespread number of persons affected with AD, scientists have undertaken the initiative to develop a cure for this devastating disease; however, it has been deemed quite challenging. A dysfunctional blood-brain barrier, with impaired ability to clear amyloid-ß from the brain, has been directly linked to the development of Alzheimer's disease. The blood-brain barrier restricts the flow of many substances into and out of the brain and serves as a selective and protective barrier to the brain. A proper functioning blood-brain barrier contributes to the maintenance and integrity of the brain. In turn, different systems and mechanisms within the blood-brain barrier are set in place to facilitate mediated passage of materials and substances between the brain and the bloodstream. In relation to AD, the mediation of amyloid-ß clearance is of great importance in maintaining the blood-brain barrier's integrity.

Investigation of Endothelial Surface Glycocalyx Components and Ultrastructure by Single Molecule Localization Microscopy: Stochastic Optical Reconstruction Microscopy (STORM).

On the luminal surface of our blood vessels, there is a thin layer called endothelial surface glycocalyx (ESG) which consists of proteoglycans, glycosaminoglycans (GAGs), and glycoproteins. The GAGs in the ESG are heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS), and sialic acid (SA). In order to play important roles in regulating vascular functions, such as being a mechanosensor and transducer for the endothelial cells (ECs) to sense the blood flow, a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall, the ESG should have an organized structure at the molecular level. Due to the limitations of conventional optical and electrical microscopy, the ultrastructure of ESG, in the order of 10 to 100 nanometers, has not been revealed until recent development of a super resolution fluorescence optical microscope, Stochastic Optical Reconstruction Microscope (STORM), which is one type of single molecule localization microscopy. This short review describes how the STORM can overcome the diffraction barrier in the conventional fluorescence microscopy to identify the chemical components of the ESG at a high spatial resolution. Examples of the organized ultrastructure of the ESG on the in vitro EC monolayer revealed by the Nikon-STORM system are given as well as how its components get lost during the onset of sepsis, a systemic inflammatory syndrome induced by bacterial infection, which demonstrate that this new technique can be applied to discover the structural and molecular mechanisms at nanometer scales in the native cellular environment for the cellular functions under normal and disease conditions.

Exocytosis of Endothelial Lysosome-Related Organelles Hair-Triggers a Patchy Loss of Glycocalyx at the Onset of Sepsis.

Sepsis is a systemic inflammatory syndrome induced by bacterial infection that can lead to multiorgan failure. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. Here, we tested a hypothesis that patchy degradation of ESG occurs early in sepsis and is a result of exocytosis of lysosome-related organelles. Time-lapse video microscopy revealed that exocytosis of Weibel-Palade bodies and secretory lysosomes occurred a few minutes after application of lipopolysaccharides to endothelial cells. Two therapeutic maneuvers, a nitric oxide intermediate, NG-hydroxy-l-arginine, and culture media conditioned by endothelial progenitor cells reduced the motility of lysosome-related organelles. Confocal and stochastic optical reconstruction microscopy confirmed the patchy loss of ESG simultaneously with the exocytosis of lysosome-related organelles and Weibel-Palade bodies in cultured endothelial cells and mouse aorta. The loss of ESG was blunted by pretreatment with NG-hydroxy-l-arginine or culture media conditioned by endothelial progenitor cells. Moreover, these treatments resulted in a significant reduction in deaths of septic mice. Our data support the hypothesis assigning to stress-induced exocytosis of these organelles the role of a hair-trigger for local degradation of ESG that initiates leukocyte infiltration, increase in vascular permeability, and partially accounts for the later rates of morbidity and mortality.

Mechanotransduction of the endothelial glycocalyx mediates nitric oxide production through activation of TRP channels.

The endothelial surface glycocalyx (ESG) is a carbohydrate-rich layer found on the vascular endothelium, serving critical functions in the mechanotransduction of blood flow-induced forces. One of the most important protective functions of the ESG is to mediate the production of nitric oxide (NO) in response to blood flow. However, the detailed mechanism underlying ESG's mechanotransduction of the production of NO has not been completely identified. Herein, using the cultured rat brain microvascular endothelial cells (bEnd.3) as a model system, we have implemented a combined atomic force and fluorescence microscopy approach to show that the ESG senses and transduces vertical mechanical stretch to produce NO. This rapid NO production is dependent on the presence of both heparan sulfate (HS) and hyaluronic acid (HA) in ESG, as the removal of HS and/or HA leads to a significant decrease in NO production. Moreover, the production of NO is dependent on the intake of Ca2+ via endothelial transient receptor potential (TRP) channels. Together, our results demonstrate the molecular mechanism of rapid production of NO in response to vertical mechanical stretch.

Inhibition of endothelial nitric oxide synthase decreases breast cancer cell MDA-MB-231 adhesion to intact microvessels under physiological flows.

Nitric oxide (NO) at different concentrations may promote or inhibit tumor growth and metastasis under various conditions. To test the hypothesis that tumor cells prefer to adhere to the locations with a higher endothelial NO production in intact microvessels under physiological flows and to further test that inhibiting NO production decreases tumor cell adhesion, we used intravital fluorescence microscopy to measure NO production and tumor cell adhesion in postcapillary venules of rat mesentery under normal and reduced flow conditions, and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, N(G)-monomethyl-l-arginine (l-NMMA). Rats (SD, 250-300 g) were anesthetized. A midline incision (∼2 inch) was made in the abdominal wall, and the mesentery was taken out from the abdominal cavity and spread over a coverslip for the measurement. An individual postcapillary venule (35-50 µm) was first loaded with 4,5-diaminofluorescein diacetate (DAF-2 DA), a fluorescent indictor for NO. Then the DAF-2 intensity was measured for 30 min under a normal or reduced flow velocity, with and without perfusion with MDA-MB-231 breast cancer cells, and in the presence of l-NMMA. We found that tumor cells prefer to adhere to the microvessel locations with a higher NO production such as curved portions. Inhibition of eNOS by l-NMMA attenuated the flow-induced NO production and reduced tumor cell adhesion. We also found that l-NMMA treatment for ∼40 min reduced microvessel permeability to albumin. Our results suggest that inhibition of eNOS is a good approach to preventing tumor cell adhesion to intact microvessels under physiological flows.

Sphingosine-1-phosphate Maintains Normal Vascular Permeability by Preserving Endothelial Surface Glycocalyx in Intact Microvessels.

OBJECTIVE: S1P was found to protect the ESG by inhibiting MMP activity-dependent shedding of ESG in cultured endothelial cell studies. We aimed to further test that S1P contributes to the maintenance of normal vascular permeability by protecting the ESG in intact microvessels. METHODS: We quantified the ESG in post-capillary venules of rat mesentery and measured the vascular permeability to albumin in the presence and absence of 1 µM S1P. We also measured permeability to albumin in the presence of MMP inhibitors and compared the measured permeability with those predicted by a transport model for the inter-endothelial cleft. RESULTS: We found that in the absence of S1P, the fluorescence intensity of the FITC-anti-HS-labeled ESG was ~10% of that in the presence of S1P, whereas the measured permeability to albumin was ~6.5-fold of that in the presence of S1P. Similar results were observed with MMP inhibition. The predictions by the mathematical model further confirmed that S1P maintains microvascular permeability by preserving ESG. CONCLUSIONS: Our results show that S1P contributes to the maintenance of normal vascular permeability by protecting the ESG in intact microvessels, consistent with parallel observation in cultured endothelial monolayers.

Sphingosine 1-phosphate induced synthesis of glycocalyx on endothelial cells.

Sphingosine 1-phosphate (S1P) protects glycocalyx against shedding, playing important roles in endothelial functions. We previously found that glycocalyx on endothelial cells (ECs) was shed after plasma protein depletion. In the present study, we investigated the role of S1P on the recovery of glycocalyx, and tested whether it is mediated by phosphoinositide 3-kinase (PI3K) pathway. After depletion of plasma protein, ECs were treated with S1P for another 6h. And then, the major components of glycocalyx including syndecan-1 with attached heparan sulfate (HS) and chondroitin sulfate (CS) on endothelial cells were detected using confocal fluorescence microscopy. Role of PI3K in the S1P-induced synthesis of glycocalyx was confirmed by using the PI3K inhibitor (LY294002). Syndecan-1 with attached HS and CS were degraded with duration of plasma protein depletion. S1P induced recovery of syndecan-1 with attached HS and CS. The PI3K inhibitor LY294002 abolished the effect of S1P on recovery of glycocalyx. Thus, S1P induced synthesis of glycocalyx on endothelial cells and it is mediated by PI3K pathway.

Temporal effects of vascular endothelial growth factor and 3,5-cyclic monophosphate on blood-brain barrier solute permeability in vivo.

To test the hypothesis that vascular endothelial growth factor (VEGF) can transiently increase the blood-brain barrier permeability, P, as for peripheral microvessels and that the elevation of 3,5-cyclic monophosphate (cAMP) levels can inhibit the VEGF-induced acute hyperpermeability, we employed multiphoton microscopy to quantify the cerebral microvessel permeability P to various-sized solutes under VEGF and cAMP treatments. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a microgrinder. Fluorescein (MW 376Da), fluorescein isothioyanate-dextran-20k (FITC-Dex-20k), FITC-Dex-70k, or Alexa Fluor 488-IgG in 1% bovine serum albumin mammalian Ringer's solution was injected into the cerebral circulation via the ipsilateral carotid artery with a syringe pump. Simultaneously, temporal images were collected from the brain parenchyma ∼100-200 µm below the pia mater. P was determined from the rate of tissue solute accumulation around individual microvessels. Exposure to 1 nM VEGF transiently increased P to 2.2, 10.5, 9.8, and 12.8 times control values, for fluorescein, Dex-20k, Dex-70k, and IgG, respectively, within 30 sec, and all returned to control levels within 2 min. After 20 min of pretreatment with 2 mM of the cAMP analog 8-bromo-cAMP, the initial increase by 1 nM VEGF was completely abolished in P of all solutes. The response pattern of P to VEGF and cAMP and the ratios of the peak to control values for rat cerebral microvessels are similar to those for rat mesenteric (peripheral) microvessels, except that the ratios are higher in P of cerebral microvessels for the intermediate and large solutes. These results imply a new approach for delivering large therapeutic agents to the brain.

Quantification of blood-brain barrier solute permeability and brain transport by multiphoton microscopy.

Development of an optimal systemic drug delivery strategy to the brain will require noninvasive or minimally invasive methods to quantify the permeability of the cerebral microvessel wall or blood-brain barrier (BBB) to various therapeutic agents and to measure their transport in the brain tissue. To address this problem, we used laser-scanning multiphoton microscopy to determine BBB permeability to solutes (P) and effective solute diffusion coefficients (Deff) in rat brain tissue 100-250 µm below the pia mater. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a microgrinder. Sodium fluorescein, fluorescein isothiocyanate (FITC)-dextrans, or Alexa Fluor 488-immunoglobulin G (IgG) in 1% bovine serum albumin (BSA) mammalian Ringer's solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of ∼3 ml/min. P and Deff were determined from the rate of tissue solute accumulation and the radial concentration gradient around individual microvessels in the brain tissue. The mean apparent permeability P values for sodium fluorescein (molecular weight (MW) 376 Da), dextran-4k, -20k, -40k, -70k, and IgG (MW ∼160 kDa) were 14.6, 6.2, 1.8, 1.4, 1.3, and 0.54 × 10-7 cm/s, respectively. These P values were not significantly different from those of rat pial microvessels for the same-sized solutes (Yuan et al., 2009, "Non-Invasive Measurement of Solute Permeability in Cerebral Microvessels of the Rat," Microvasc. Res., 77(2), pp. 166-73), except for the small solute sodium fluorescein, suggesting that pial microvessels can be a good model for studying BBB transport of relatively large solutes. The mean Deff values were 33.2, 4.4, 1.3, 0.89, 0.59, and 0.47 × 10-7 cm2/s, respectively, for sodium fluorescein, dextran-4k, -20k, -40k, -70k, and IgG. The corresponding mean ratio of Deff to the free diffusion coefficient Dfree, Deff/Dfree, were 0.46, 0.19, 0.12, 0.12, 0.11, and 0.11 for these solutes. While there is a significant difference in Deff/Dfree between small (e.g., sodium fluorescein) and larger solutes, there is no significant difference in Deff/Dfree between solutes with molecular weights from 20,000 to 160,000 Da, suggesting that the relative resistance of the brain tissue to macromolecular solutes is similar over a wide size range. The quantitative transport parameters measured from this study can be used to develop better strategies for brain drug delivery.

Angiogenesis and Microvascular Permeability.

Angiogenesis, the formation of new blood microvessels, is a necessary physiological process for tissue generation and repair. Sufficient blood supply to the tissue is dependent on microvascular density, while the material exchange between the circulating blood and the surrounding tissue is controlled by microvascular permeability. We thus begin this article by reviewing the key signaling factors, particularly vascular endothelial growth factor (VEGF), which regulates both angiogenesis and microvascular permeability. We then review the role of angiogenesis in tissue growth (bone regeneration) and wound healing. Finally, we review angiogenesis as a pathological process in tumorigenesis, intraplaque hemorrhage, cerebral microhemorrhage, pulmonary fibrosis, and hepatic fibrosis. Since the glycocalyx is important for both angiogenesis and microvascular permeability, we highlight the role of the glycocalyx in regulating the interaction between tumor cells and endothelial cells (ECs) and VEGF-containing exosome release and uptake by tumor-associated ECs, all of which contribute to tumorigenesis and metastasis.

Heparan Sulfate Modulation Affects Breast Cancer Cell Adhesion and Transmigration across In Vitro Blood-Brain Barrier.

The disruption of endothelial heparan sulfate (HS) is an early event in tumor cell metastasis across vascular barriers, and the reinforcement of endothelial HS reduces tumor cell adhesion to endothelium. Our recent study showed that while vascular endothelial growth factor (VEGF) greatly reduces HS at an in vitro blood-brain barrier (BBB) formed by human cerebral microvascular endothelial cells (hCMECs), it significantly enhances HS on a breast cancer cell, MDA-MB-231 (MB231). Here, we tested that this differential effect of VEGF on the HS favors MB231 adhesion and transmigration. We also tested if agents that enhance endothelial HS may affect the HS of MB231 and reduce its adhesion and transmigration. To test these hypotheses, we generated an in vitro BBB by culturing hCMECs on either a glass-bottom dish or a Transwell filter. We first quantified the HS of the BBB and MB231 after treatment with VEGF and endothelial HS-enhancing agents and then quantified the adhesion and transmigration of MB231 across the BBB after pretreatment with these agents. Our results demonstrated that the reduced/enhanced BBB HS and enhanced/reduced MB231 HS increase/decrease MB231 adhesion to and transmigration across the BBB. Our findings suggest a therapeutic intervention by targeting the HS-mediated breast cancer brain metastasis.

Inhibition of Abl Kinase by Imatinib Can Rescue the Compromised Barrier Function of 22q11.2DS Patient-iPSC-Derived Blood-Brain Barriers.

We have previously established that the integrity of the induced blood-brain barrier (iBBB) formed by brain microvascular endothelial cells derived from the iPSC of 22q11.2 DS (22q11.2 Deletion Syndrome, also called DiGeorge Syndrome) patients is compromised. We tested the possibility that the haploinsufficiency of CRKL, a gene within the 22q11.2 DS deletion region, contributes to the deficit. The CRKL is a major substrate of the Abl tyrosine kinase, and the Abl/CRKL signaling pathway is critical for endothelial barrier functions. Imatinib, an FDA-approved drug, inhibits Abl kinase and has been used to treat various disorders involving vascular leakages. To test if imatinib can restore the compromised iBBB, we treated the patient's iBBB with imatinib. After treatment, both trans-endothelial electrical resistance and solute permeability returned to comparable levels of the control iBBB. Correspondingly, changes in tight junctions and endothelial glycocalyx of the iBBB were also restored. Western blotting showed that imatinib increased the level of active forms of the CRKL protein. A transcriptome study revealed that imatinib up-regulated genes in the signaling pathways responsible for the protein modification process and down-regulated those for cell cycling. The KEGG pathway analysis further suggested that imatinib improved the gene expression of the CRKL signaling pathway and tight junctions, which agrees with our expectations and the observations at protein levels. Our results indicate that the 22q11.2DS iBBB is at least partially caused by the haploinsufficiency of CRKL, which can be rescued by imatinib via its effects on the Abl/CRKL signaling pathway. Our findings uncover a novel disease mechanism associated with 22q11.2DS.

Quantification of the endothelial surface glycocalyx on rat and mouse blood vessels.

The glycocalyx on the surface of endothelium lining blood vessel walls modulates vascular barrier function, cell adhesion and also serves as a mechano-sensor for blood flow. Reduction of glycocalyx has been reported in many diseases including atherosclerosis, inflammation, myocardial edema, and diabetes. The surface glycocalyx layer (SGL) is composed of proteoglycans and glycosaminoglycans, of which heparan sulfate is one of the most abundant. To quantify the SGL thickness on the microvessels of rat mesentery and mouse cremaster muscle in situ, we applied a single vessel cannulation and perfusion technique to directly inject FITC-anti-heparan sulfate into a group of microvessels for immuno-labeling the SGL. We also used anti-heparan sulfate for immuno-labeling the SGL on rat and mouse aortas ex vivo. High resolution confocal microscopy revealed that the thickness of the SGL on rat mesenteric capillaries and post-capillary venules is 0.9±0.1 µm and 1.2±0.3 µm, respectively; while the thickness of the SGL on mouse cremaster muscle capillaries and post-capillary venules is 1.5±0.1 µm and 1.5±0.2 µm, respectively. Surprisingly, there was no detectable SGL in either rat mesenteric or mouse cremaster muscle arterioles. The SGL thickness is 2.5±0.1 µm and 2.1±0.2 µm respectively, on rat and mouse aorta. In addition, we observed that the SGL is continuously and evenly distributed on the aorta wall but not on the microvessel wall.

Effect of wall compliance and permeability on blood-flow rate in counter-current microvessels formed from anastomosis during tumor-induced angiogenesis.

Tumor blood-flow is inhomogeneous because of heterogeneity in tumor vasculature, vessel-wall leakiness, and compliance. Experimental studies have shown that normalization of tumor vasculature by antiangiogenic therapy can improve tumor microcirculation and enhance the delivery of therapeutic agents to tumors. To elucidate the quantitative relationship between the vessel-wall compliance and permeability and the blood-flow rate in the microvessels of the tumor tissue, the tumor tissue with the normalized vasculature, and the normal tissue, we developed a transport model to simultaneously predict the interstitial fluid pressure (IFP), interstitial fluid velocity (IFV) and the blood-flow rate in a counter-current microvessel loop, which occurs from anastomosis in tumor-induced angiogenesis during tumor growth. Our model predicts that although the vessel-wall leakiness greatly affects the IFP and IFV, it has a negligible effect on the intravascular driving force (pressure gradient) for both rigid and compliant vessels, and thus a negligible effect on the blood-flow rate if the vessel wall is rigid. In contrast, the wall compliance contributes moderately to the IFP and IFV, but significantly to the vessel radius and to the blood-flow rate. However, the combined effects of vessel leakiness and compliance can increase IFP, which leads to a partial collapse in the blood vessels and an increase in the flow resistance. Furthermore, our model predictions speculate a new approach for enhancing drug delivery to tumor by modulating the vessel-wall compliance in addition to reducing the vessel-wall leakiness and normalizing the vessel density.