The blood vessels of the central nervous (CNS) system form a tight, protective blood-brain barrier (BBB). This barrier is essential for healthy CNS function but also pose... More
The blood vessels of the central nervous (CNS) system form a tight, protective blood-brain barrier (BBB). This barrier is essential for healthy CNS function but also poses a hurdle in the treatment of increasingly common neurological disorders. Additionally, BBB dysfunction is a hallmark of many neurological diseases, further emphasizing a need for a better understanding of BBB function in health and disease. We present a human self-assembling 3D model of the BBB in a microfluidic cell culture platform that allows culture of 48 models in parallel on one tissue culture plate. Human brain microvascular endothelial cells, pericytes, and astrocytes form highly reproducible BBB vascular networks under unidirectional perfusion and remain viable for a minimum of 14 days. Immunostaining reveals close cell-cell interactions with pericytes and astrocyte endfeet in direct contact with the brain microvasculature. Compared to endothelial monocultures, co-cultures with astrocytes or pericytes result in improved barrier function, lower vessel diameters, increased branching, and alignment of the vessels in the direction of fluid flow. These results were most pronounced in tri-cultures containing all three cell types. Unlike similar models previously reported, this brain microvasculature model allows for unidirectional perfusion without the need for pumps and syringes. Combined with its high-throughput nature, this feature renders the model suitable for studies of BBB function in health and disease, and assessment of potential BBB restorative therapies. Less
Longitudinal live cell imaging is valuable for characterizing dynamic morphological and phenotypic changes in biological systems. How-ever, conventional approaches rely o... More
Longitudinal live cell imaging is valuable for characterizing dynamic morphological and phenotypic changes in biological systems. How-ever, conventional approaches rely on manual microscope operation, which is labor-intensive, limits imaging frequency, and disrupts the cellular environment. These constraints reduce scalability, increase experimental variability, and restrict both the duration and temporal resolution of continuous imaging. Although auto-mated imaging platforms partially address these limitations, existing solutions are often constrained by the cost, footprint, and inflexibility of in-incubator microscopes or stage-top incubators. Here, we present an automated in-incubator epifluorescence micro-scope designed for long-term operation. The system features a modular architecture with optional multi-fluorescence imaging, automated plate scanning, configurable light sources, and compatibility with multiple plate formats, including integration with fluidic automation devices. By positioning the light sources and control electronics outside the incubator, the platform improves thermal stability and long-term operational reliability. This approach enables continuous, high-frequency imaging over extended durations, providing a source of rich data for quantifying time-dependent tissue phenotypes, morphological remodeling, and transient biological processes. Less
Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) which leads to infiltration of blood cells, an inflammatory response, and neuronal... More
Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) which leads to infiltration of blood cells, an inflammatory response, and neuronal cell death, resulting spinal cord secondary damage. Retinoic acid (RA) has a neuroprotective effect in both ischemic brain injury and SCI, however the relationship between BSCB disruption and RA in SCI is still unclear. In this study, we demonstrated that autophagy and ER stress are involved in the protective effect of RA on the BSCB. RA attenuated BSCB permeability and decreased the loss of tight junction (TJ) molecules such as P120, β-catenin, Occludin and Claudin5 after injury in vivo as well as in Brain Microvascular Endothelial Cells (BMECs). Moreover, RA administration improved functional recovery in the rat model of SCI. RA inhibited the expression of CHOP and caspase-12 by induction of autophagic flux. However, RA had no significant effect on protein expression of GRP78 and PDI. Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB via exacerbated ER stress and subsequent loss of tight junctions. Taken together, the neuroprotective role of RA in recovery from SCI is related to prevention of of BSCB disruption via the activation of autophagic flux and the inhibition of ER stress-induced cell apoptosis. These findings lay the groundwork for future translational studies of RA for CNS diseases, especially those related to BSCB disruption. Less
The homeostasis of the central nervous system is maintained by the blood–brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angi... More
The homeostasis of the central nervous system is maintained by the blood–brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angiogenesis, vascular stability, vascular permeability and lymphatic integrity. However, the precise role of angiopoietin/Tie2 signaling at the BBB remains unclear. We investigated the influence of Ang-2 on BBB permeability in wild-type and gain-of-function (GOF) mice and demonstrated an increase in permeability by Ang-2, both in vitro and in vivo. Expression analysis of brain endothelial cells from Ang-2 GOF mice showed a downregulation of tight/adherens junction molecules and increased caveolin-1, a vesicular permeability-related molecule. Immunohistochemistry revealed reduced pericyte coverage in Ang-2 GOF mice that was supported by electron microscopy analyses, which demonstrated defective intra-endothelial junctions with increased vesicles and decreased/disrupted glycocalyx. These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes. In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated. In mice, Ang-2 GOF resulted in increased infarct sizes and vessel permeability upon experimental stroke, implicating a role of Ang-2 in stroke pathophysiology. Increased permeability and stroke size were rescued by activation of Tie2 signaling using a vascular endothelial protein tyrosine phosphatase inhibitor and were independent of VE-cadherin phosphorylation. We thus identified Ang-2 as an endothelial cell-derived regulator of BBB permeability. We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema. Less
We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4). However, Gram-negative pathogen-associat... More
We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4). However, Gram-negative pathogen-associated molecular patterns include not only LPS but also peptidoglycan (PGN). Given that the presence of co-purified PGN in the LPS preparation previously used could not be ruled out, we decided to analyse the expression of the intracellular PGN receptors NOD1 and NOD2 in HBP and compare the responses to their cognate agonists and ultrapure LPS. Our findings show for the first time that NOD1 is expressed in pericytes, whereas NOD2 expression is barely detectable. The NOD1 agonist C12-iE-DAP induced IL6 and IL8 gene expression by pericytes as well as release of cytokines into culture supernatant. Moreover, we demonstrated the synergistic effects of NOD1 and TLR4 agonists on the induction of IL8. Using NOD1 silencing in HBP, we showed a requirement for C12-iE-DAP-dependent signalling. Finally, we could discriminate NOD1 and TLR4 pathways in pericytes by pharmacological targeting of RIPK2, a kinase involved in NOD1 but not in TLR4 signalling cascade. p38 MAPK and NF-κB appear to be downstream mediators in the NOD1 pathway. In summary, these results indicate that pericytes can sense Gram-negative bacterial products by both NOD1 and TLR4 receptors, acting through distinct pathways. This provides new insight about how brain pericytes participate in the inflammatory response and may have implications for disease management. Keywords: NOD1; TLR4; inflammation; lipopolysaccharide; peptidoglycan; pericyte; vascular biology. Less
Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have g... More
Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM-MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion-induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM-MSCs. Seventy mice were pre-treated with BM-MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro-vascular endothelial cells (HPMVECs) were pre-conditioned with BM-MSCs by oxygen-glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI-treated mice, administration of BM-MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD-treated HPMVECs, co-culture with BM-MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM-MSCs decreased the level of PI3K class I and p-Akt while the expression of PI3K class III was increased. Finally, BM-MSCs-induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM-MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM-MSCs and will help to develop new cell-based therapeutic strategies in lung injury. Less
Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progres... More
Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease. Less
ABSTRACT: The effects of mechanical cues on cell behaviors in 3D remain difficult to characterize as the ability to tune hydrogel mechanics often requires changes in the ... More
ABSTRACT: The effects of mechanical cues on cell behaviors in 3D remain difficult to characterize as the ability to tune hydrogel mechanics often requires changes in the polymer density, potentially altering the material’s biochemical and physical characteristics. Additionally, with most PEG diacrylate (PEGDA) hydrogels, forming materials with compressive moduli less than ∼10 kPa has been virtually impossible. Here, we present a new method of controlling the mechanical properties of PEGDA hydrogels independent of polymer chain density through the incorporation of additional vinyl group moieties that interfere with the cross-linking of the network. This modification can tune hydrogel mechanics in a concentration dependent manner from <1 to 17 kPa, a more physiologically relevant range than previously possible with PEG-based hydrogels, without altering the hydrogel’s degradation and permeability. Across this range of mechanical properties, endothelial cells (ECs) encapsulated within MMP-2/MMP-9 degradable hydrogels with RGDS adhesive peptides revealed increased cell spreading as hydrogel stiffness decreased in contrast to behavior typically observed for cells on 2D surfaces. EC-pericyte cocultures exhibited vessel-like networks within 3 days in highly compliant hydrogels as compared to a week in stiffer hydrogels. These vessel networks persisted for at least 4 weeks and deposited laminin and collagen IV perivascularly. These results indicate that EC morphogenesis can be regulated using mechanical cues in 3D. Furthermore, controlling hydrogel compliance independent of density allows for the attainment of highly compliant mechanical regimes in materials that can act as customizable cell microenvironments. KEYWORDS: vasculogenesis, tissue engineering, biomimetic material, PEG hydrogel Less
Primary Familial Brain Calcification (PFBC), a neurodegenerative disease characterized by progressive pericapillary calcifications, has recently been linked to heterozygo... More
Primary Familial Brain Calcification (PFBC), a neurodegenerative disease characterized by progressive pericapillary calcifications, has recently been linked to heterozygous mutations in PDGFB and PDGFRB genes. Here, we functionally analyzed several of these mutations in vitro. All six analyzed PDGFB mutations led to complete loss of PDGF-B function either through abolished protein synthesis or through defective binding and/or stimulation of PDGF-Rβ. The three analyzed PDGFRB mutations had more diverse consequences. Whereas PDGF-Rβ autophosphorylation was almost totally abolished in the PDGFRB L658P mutation, the two sporadic PDGFRB mutations R987W and E1071V caused reductions in protein levels and specific changes in the intensity and kinetics of PLCγ activation, respectively. Since at least some of the PDGFB mutations were predicted to act through haploinsufficiency, we explored the consequences of reduced Pdgfb or Pdgfrb transcript and protein levels in mice. Heterozygous Pdgfb or Pdgfrb knockouts, as well as double Pdgfb+/-;Pdgfrb+/- mice did not develop brain calcification, nor did Pdgfrbredeye/redeye mice, which show a 90% reduction of PDGFRβ protein levels. In contrast, Pdgfbret/ret mice, which have altered tissue distribution of PDGF-B protein due to loss of a proteoglycan binding motif, developed brain calcifications. We also determined pericyte coverage in calcification-prone and non-calcification-prone brain regions in Pdgfbret/ret mice. Surprisingly and contrary to our hypothesis, we found that the calcification-prone brain regions in Pdgfbret/ret mice model had a higher pericyte coverage and a more intact blood-brain barrier (BBB) compared to non-calcification-prone brain regions. While our findings provide clear evidence that loss-of-function mutations in PDGFB or PDGFRB cause PFBC, they also demonstrate species differences in the threshold levels of PDGF-B/PDGF-Rβ signaling that protect against small-vessel calcification in the brain. They further implicate region-specific susceptibility factor(s) in PFBC pathogenesis that are distinct from pericyte and BBB deficiency. Less
Retinopathy of prematurity causes visual impairment due to destructive neoangiogenesis after degeneration of the retinal microvasculature. This study was aimed at analyzi... More
Retinopathy of prematurity causes visual impairment due to destructive neoangiogenesis after degeneration of the retinal microvasculature. This study was aimed at analyzing whether local delivery of Semaphorin-3C (Sema3C) suppresses pathological retinal angiogenesis. Sema3C exerted potent inhibiting effects in cellular models of angiogenesis. In an endothelial cell xenotransplantation assay, Sema3C acted primarily on immature microvessels by inducing endothelial cell apoptosis. Intravitreal administration of recombinant Sema3C disrupted endothelial tip cell formation and cell-cell contacts, which led to decreased vascular bed expansion and vessel branching in the growing retinal vasculature of newborn mice, while not affecting mature vessels in the adult retina. Sema3C administration strongly inhibited the formation of pathological pre-retinal vascular tufts during oxygen-induced retinopathy. Mechanistically, Sema3C signaled through the receptors Neuropilin-1 and PlexinD1, which were strongly expressed on vascular tufts, induced VE-cadherin internalization, and abrogated vascular endothelial growth factor (VEGF)-induced activation of the kinases AKT, FAK, and p38MAPK. This disrupted endothelial cell junctions, focal adhesions, and cytoskeleton assembly resulted in decreased cell migration and survival. Thus, this study identified Sema3C as a potent and selective inhibitor of pathological retinal angiogenesis. Keywords: Sema3C; angiogenesis; retinopathy of prematurity; semaphorin. Less
Rationale: Pericytes are perivascular cells localized to capillaries that promote vessel maturation and their absence can contribute to vessel loss. Whether impaired peri... More
Rationale: Pericytes are perivascular cells localized to capillaries that promote vessel maturation and their absence can contribute to vessel loss. Whether impaired pericyte interaction with endothelial cells contributes to small vessel loss in pulmonary arterial hypertension (PAH) is unclear. Objective: To measure the ability of PAH pericytes to associate with pulmonary microvascular endothelial cells (PMVECs) in vitro and in vivo. Methods and Results: Using 3G5 specific IgG-coated magnetic beads, we isolated pericytes from lungs of healthy and PAH patients followed by lineage validation. When seeded with healthy PMVECs, PAH pericytes failed to associate with endothelial tubes, resulting in smaller vascular networks compared to those seen with healthy pericytes. Following demonstration of abnormal polarization towards endothelium via live imaging and wound-healing studies, we screened PAH pericytes for abnormalities in the Wnt/planar cell polarity (PCP) pathway, which has been shown to regulate cell motility and polarity in the pulmonary vasculature. We found that PAH pericytes have reduced expression of frizzled 7 (Fzd7) and cdc42, two genes critical for Wnt/PCP activation. Simultaneous knockdown of Fzd7 and cdc42 in healthy pericytes resulted in reduced motility and polarization towards PMVECs both in vitro and in a murine model of angiogenesis, whereas restoration of both genes in PAH pericytes resulted in improved endothelial-pericyte association and larger vascular networks. Conclusions: These studies suggest that the motility and polarity of pericytes during pulmonary angiogenesis is regulated by Wnt/PCP activation. Therapies targeting Wnt/PCP in pericytes could help prevent vessel loss in PAH. Less
Purpose The blood-brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain end... More
Purpose The blood-brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transporting drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule 1 (ICAM-1), to transport drug carriers into and across BBB models. Methods Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures. Results ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB. Conclusions CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier. Keywords: ICAM-1-targeted nanocarriers, clathrin- and caveolae-independent transport, CAM-mediated endocytosis, blood-brain barrier transport, brain endothelial and subendothelial cell layers Less
The NG2 proteoglycan stimulates the proliferation and migration of various immature cell types, including pericytes. However, the role of NG2 in mediating pericyte/endoth... More
The NG2 proteoglycan stimulates the proliferation and migration of various immature cell types, including pericytes. However, the role of NG2 in mediating pericyte/endothelial cell interaction has been less clear. In this study, we show that pericyte-specific NG2 ablation causes several structural deficits in blood vessels in intracranial B16F10 melanomas, including decreased pericyte ensheathment of endothelial cells, diminished formation of endothelial junctions, and reduced assembly of the vascular basal lamina. These deficits result in decreased tumor vessel patency, increased vessel leakiness, and increased intratumoral hypoxia. NG2-dependent mechanisms of pericyte interaction with endothelial cells are further explored in pericyte/endothelial cell co-cultures. siRNA-mediated NG2 knockdown in pericytes leads to reduced formation of pericyte/endothelial networks, reduced formation of ZO-1 positive endothelial cell junctions, and increased permeability of endothelial cell monolayers. We also show that NG2 knockdown results in loss of β1 integrin activation in endothelial cells, revealing a mechanism for NG2-dependent cross talk between pericytes and endothelial cells. Less
Diabetic retinopathy is one of the main microvascular complications of diabetes and remains one of the leading causes of blindness worldwide. Recent studies have revealed... More
Diabetic retinopathy is one of the main microvascular complications of diabetes and remains one of the leading causes of blindness worldwide. Recent studies have revealed an important role of inflammatory and proangiogenic high mobility group 1 (HMGB-1) cytokine in diabetic retinopathy. To elucidate cellular mechanisms of HMGB-1 activity in the retina, we performed this study. The histological features of diabetic retinopathy include loss of blood-vessel pericytes and endothelial cells, as well as abnormal new blood vessel growth. To establish the role of HMGB-1 in vulnerability of endothelial cells and pericytes, cultures of these cells, or co-cultures with glial cells, were treated with HMGB-1 and assessed for survival after 24 hours. The expression levels of the cytokines, chemokines, and cell adhesion molecules in glial and endothelial cells were tested by quantitative RT-PCR to evaluate changes in these cells after HMGB-1 treatment. Animal models of neovascularization were also used to study the role of HMGB-1 in the retina. We report that pericyte death is mediated by HMGB-1-induced cytotoxic activity of glial cells, while HMGB-1 can directly mediate death of endothelial cells. We also found that HMGB-1 affects endothelial cell activity. However, we did not observe a difference in the levels of neovascularization between HMGB-1-treated eyes compared to the control eyes, nor in the levels of proangiogenic cytokine VEGF-A expression between glial cells treated with HMGB-1 and control cells. Our data also indicate that HMGB-1 is not involved in retinal neovascularization in the oxygen-induced retinopathy model. Thus, our data suggest that retinal pericyte and endothelial injury and death in diabetic retinopathy may be due to HMGB-1-induced cytotoxic activity of glial cells as well as the direct effect of HMGB-1 on endothelial cells. At the same time, our findings indicate that HMGB-1 plays an insignificant role in retinal and choroidal neovascularization. Less
Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human mode... More
Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge, we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons derived from renewable cell sources. First, retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs, particularly through adherens junction, tight junction and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (~5,000 Ωxcm2). Overall, this scalable human BBB model may enable a wide range of neuroscience studies. Less
Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human mode... More
Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge, we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons derived from renewable cell sources. First, retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs, particularly through adherens junction, tight junction and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (~5,000 Ωxcm2). Overall, this scalable human BBB model may enable a wide range of neuroscience studies. Less
Diabetic retinopathy is one of the main microvascular complications of diabetes and remains one of the leading causes of blindness worldwide. Recent studies have revealed... More
Diabetic retinopathy is one of the main microvascular complications of diabetes and remains one of the leading causes of blindness worldwide. Recent studies have revealed an important role of inflammatory and proangiogenic high mobility group 1 (HMGB-1) cytokine in diabetic retinopathy. To elucidate cellular mechanisms of HMGB-1 activity in the retina, we performed this study. The histological features of diabetic retinopathy include loss of blood-vessel pericytes and endothelial cells, as well as abnormal new blood vessel growth. To establish the role of HMGB-1 in vulnerability of endothelial cells and pericytes, cultures of these cells, or co-cultures with glial cells, were treated with HMGB-1 and assessed for survival after 24 hours. The expression levels of the cytokines, chemokines, and cell adhesion molecules in glial and endothelial cells were tested by quantitative RT-PCR to evaluate changes in these cells after HMGB-1 treatment. Animal models of neovascularization were also used to study the role of HMGB-1 in the retina. We report that pericyte death is mediated by HMGB-1-induced cytotoxic activity of glial cells, while HMGB-1 can directly mediate death of endothelial cells. We also found that HMGB-1 affects endothelial cell activity. However, we did not observe a difference in the levels of neovascularization between HMGB-1-treated eyes compared to the control eyes, nor in the levels of proangiogenic cytokine VEGF-A expression between glial cells treated with HMGB-1 and control cells. Our data also indicate that HMGB-1 is not involved in retinal neovascularization in the oxygen-induced retinopathy model. Thus, our data suggest that retinal pericyte and endothelial injury and death in diabetic retinopathy may be due to HMGB-1-induced cytotoxic activity of glial cells as well as the direct effect of HMGB-1 on endothelial cells. At the same time, our findings indicate that HMGB-1 plays an insignificant role in retinal and choroidal neovascularization. Less
The NG2 proteoglycan stimulates the proliferation and migration of various immature cell types, including pericytes. However, the role of NG2 in mediating pericyte/endoth... More
The NG2 proteoglycan stimulates the proliferation and migration of various immature cell types, including pericytes. However, the role of NG2 in mediating pericyte/endothelial cell interaction has been less clear. In this study, we show that pericyte-specific NG2 ablation causes several structural deficits in blood vessels in intracranial B16F10 melanomas, including decreased pericyte ensheathment of endothelial cells, diminished formation of endothelial junctions, and reduced assembly of the vascular basal lamina. These deficits result in decreased tumor vessel patency, increased vessel leakiness, and increased intratumoral hypoxia. NG2-dependent mechanisms of pericyte interaction with endothelial cells are further explored in pericyte/endothelial cell co-cultures. siRNA-mediated NG2 knockdown in pericytes leads to reduced formation of pericyte/endothelial networks, reduced formation of ZO-1 positive endothelial cell junctions, and increased permeability of endothelial cell monolayers. We also show that NG2 knockdown results in loss of β1 integrin activation in endothelial cells, revealing a mechanism for NG2-dependent cross talk between pericytes and endothelial cells. Less
In postmenopausal women, estrogen (E2) deficiencies are frequently associated with higher risk of intracranial hemorrhage, increased incidence of stroke, cerebral aneurys... More
In postmenopausal women, estrogen (E2) deficiencies are frequently associated with higher risk of intracranial hemorrhage, increased incidence of stroke, cerebral aneurysm, and decline in cognitive abilities. In younger postpartum women and those using oral contraceptives, perturbations in E2 are associated with higher risk of cerebral venous thrombosis. A number of serious intracranial pathologic conditions linked to E2 deficiencies, such as dural sinus thrombosis, dural fistulae, non-parenchymal intracranial hemorrhages, migraines, and spontaneous cerebrospinal fluid leaks, involve the vessels not of the brain itself, but of the outer fibrous membrane of the brain, the dura mater (DM). The pathogenesis of these disorders remains mysterious and how estrogen regulates structural and functional integrity of DM vasculature is largely unknown. Here, we demonstrate that post ovariectomy (OVX) DM vascular remodeling is manifested by microvessel destabilization, capillary rarefaction, increased vascular permeability, and aberrant angio-architecture, and is the result of disrupted E2-regulated PDGF-BB signaling within dura microvasculature. These changes, associated with the reduction in systemic PDGF-BB levels, are not corrected by a flat-dose E2 hormone replacement therapy (HRT), but are largely prevented using HRT schedules mimicking physiological E2 fluctuations. We demonstrate that 1) E2 regulates PDGF-BB production by endothelial cells in a dose-dependent manner and 2) optimization of PDGF-BB levels and induction of robust PDGF-mediated endothelial cell-vascular pericyte interactions require high (estrous) E2 concentrations. We conclude that high (estrous) levels of E2 are important in controlling PDGF-mediated crosstalk between endothelial cells and pericytes, a fundamental mechanism governing microvessel stability and essential for preserving intracranial homeostasis. Less
In postmenopausal women, estrogen (E2) deficiencies are frequently associated with higher risk of intracranial hemorrhage, increased incidence of stroke, cerebral aneurys... More
In postmenopausal women, estrogen (E2) deficiencies are frequently associated with higher risk of intracranial hemorrhage, increased incidence of stroke, cerebral aneurysm, and decline in cognitive abilities. In younger postpartum women and those using oral contraceptives, perturbations in E2 are associated with higher risk of cerebral venous thrombosis. A number of serious intracranial pathologic conditions linked to E2 deficiencies, such as dural sinus thrombosis, dural fistulae, non-parenchymal intracranial hemorrhages, migraines, and spontaneous cerebrospinal fluid leaks, involve the vessels not of the brain itself, but of the outer fibrous membrane of the brain, the dura mater (DM). The pathogenesis of these disorders remains mysterious and how estrogen regulates structural and functional integrity of DM vasculature is largely unknown. Here, we demonstrate that post ovariectomy (OVX) DM vascular remodeling is manifested by microvessel destabilization, capillary rarefaction, increased vascular permeability, and aberrant angio-architecture, and is the result of disrupted E2-regulated PDGF-BB signaling within dura microvasculature. These changes, associated with the reduction in systemic PDGF-BB levels, are not corrected by a flat-dose E2 hormone replacement therapy (HRT), but are largely prevented using HRT schedules mimicking physiological E2 fluctuations. We demonstrate that 1) E2 regulates PDGF-BB production by endothelial cells in a dose-dependent manner and 2) optimization of PDGF-BB levels and induction of robust PDGF-mediated endothelial cell-vascular pericyte interactions require high (estrous) E2 concentrations. We conclude that high (estrous) levels of E2 are important in controlling PDGF-mediated crosstalk between endothelial cells and pericytes, a fundamental mechanism governing microvessel stability and essential for preserving intracranial homeostasis. Less
Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the d... More
Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold. Less
The blood brain barrier (BBB) has evolved unique characteristics such as dense coverage of the endothelial cells by pericytes and interactions with astrocytes through per... More
The blood brain barrier (BBB) has evolved unique characteristics such as dense coverage of the endothelial cells by pericytes and interactions with astrocytes through perivascular endfeet. We study BBB formation in a 3-dimensional multicellular spheroid system of human primary brain endothelial cells (hpBECs), primary pericytes (hpPs) and primary astrocytes (hpAs). We show for the first time that hpBECs, hpPs and hpAs spontaneously self-organize into a defined multicellular structure which recapitulates the complex arrangement of the individual cell types in the BBB structure. Pericytes play a crucial role mediating the interaction between hpBECs and hpAs. This process is not dependent on a scaffold support demonstrating that formation and cellular architecture of the BBB is intrinsically programmed within each specific cell type. In a matrigel setup the hpBECs, hpPs and hpAs also undergo self-arrangement to form endothelial tube-like structures tightly covered by hpPs and loosely attached hpAs mainly at the junctions. Less
Objective: Cell-mediated immune responses in peripheral tissues begin with T cell infiltration through endothelial cell (EC)-microvessels and accumulation in the perivasc... More
Objective: Cell-mediated immune responses in peripheral tissues begin with T cell infiltration through endothelial cell (EC)-microvessels and accumulation in the perivascular space occupied by pericytes (PC). Here we investigate how human T cells interact with PC. Methods and Results: We compared human placental PC with autologous umbilical vein EC. Cultured PC express lower levels of MHC and positive costimulatory molecules but higher levels of negative costimulatory molecules than do EC. Unlike EC, IFN-γ-treated MHC class II positive PC (PC+) cannot stimulate resting allogeneic CD4 T cell proliferation or cytokine production. Instead, coculture of resting CD4 T cells with PC+ induces CD25 expression and renders T cells unresponsive to restimulation by EC+ from the same donor. PC cultured across a transwell decrease alloreactive CD4 T cell proliferation to EC+, an effect enhanced by pre-treatment of PC with IFN-γ and partially reversed by IL-10 and TGF-β neutralization, but do not induce anergy. Conclusions: Human placental PC are poorly immunogenic and negatively regulate CD4 T cell responses through contact-dependent and -independent mechanisms. Keywords: pericytes, endothelial cells, alloresponse, T cell anergy, immune modulation Less
Neurovascular injury comprises a wide spectrum of pathophysiology that underlies the progression of brain injury after cerebral ischemia. Recently, it has been shown that... More
Neurovascular injury comprises a wide spectrum of pathophysiology that underlies the progression of brain injury after cerebral ischemia. Recently, it has been shown that activation of the integrin-associated protein CD47 mediates the development of blood-brain barrier injury and edema after cerebral ischemia. However, the mechanisms that mediate these complex neurovascular effects of CD47 remain to be elucidated. Here, we compare the effects of CD47 signaling in brain endothelial cells, astrocytes, and pericytes. Exposure to 4N1 K, a specific CD47-activating peptide derived from the major CD47 ligand thrombospondin-1, upregulated two major neurovascular mediators, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9), in brain endothelial cells and astrocytes. No changes were detected in pericytes. These findings may provide a potential mechanism for CD47-induced changes in blood-brain barrier homeostasis, and further suggest that CD47 may be a relevant neurovascular target in stroke. Less
Recently, we reported a novel system whereby human pericytes are recruited to endothelial cell (EC)-lined tubes in 3-dimensional (3D) extracellular matrices to stimulate ... More
Recently, we reported a novel system whereby human pericytes are recruited to endothelial cell (EC)-lined tubes in 3-dimensional (3D) extracellular matrices to stimulate vascular maturation including basement membrane matrix assembly. Through the use of this serum-free, defined system, we demonstrate that pericyte motility within 3D collagen matrices is dependent on the copresence of ECs. Using either soluble receptor traps consisting of the extracellular ligand-binding domains of platelet-derived growth factor receptor β, epidermal growth factor receptor (EGFR), and ErbB4 receptors or blocking antibodies directed to platelet-derived growth factor (PDGF)-BB, or heparin-binding EGF-like growth factor (HB-EGF), we show that both of these EC-derived ligands are required to control pericyte motility, proliferation, and recruitment along the EC tube ablumenal surface. Blockade of pericyte recruitment causes a lack of basement membrane matrix deposition and, concomitantly, increased vessel widths. Combined inhibition of PDGF-BB and HB-EGF-induced signaling in quail embryos leads to reduced pericyte recruitment to EC tubes, decreased basement membrane matrix deposition, increased vessel widths, and vascular hemorrhage phenotypes in vivo, in support of our findings in vitro. In conclusion, we report a dual role for EC-derived PDGF-BB and HB-EGF in controlling pericyte recruitment to EC-lined tubes during developmental vascularization events. Less
Purpose: Recent evidence suggests that at least some sarcomas arise through aberrant differentiation of mesenchymal stromal cells (MSCs), but MSCs have never been isolate... More
Purpose: Recent evidence suggests that at least some sarcomas arise through aberrant differentiation of mesenchymal stromal cells (MSCs), but MSCs have never been isolated directly from human sarcoma specimens.Experimental design: We examined human sarcoma cell lines and primary adherent cultures derived from human sarcoma surgical samples for features of MSCs. We further characterized primary cultures as either benign or malignant by the presence of tumor-defining genetic lesions and tumor formation in immunocompromised mice. Results: We show that a dedifferentiated liposarcoma cell line DDLS8817 posesses fat, bone, and cartilage trilineage differentiation potential characteristic of MSCs. Primary sarcoma cultures have the morphology, surface immunophenotype, and differentiation potential characteristic of MSCs. Surprisingly, many of these cultures are benign, as they do not form tumors in mice and lack sarcoma-defining genetic lesions. Consistent with the recently proposed pericyte origin of MSCs in normal human tissues, sarcoma-derived benign MSCs (SDBMSCs) express markers of pericytes and cooperate with endothelial cells in tube formation assays. In human sarcoma specimens, a subset of CD146-positive microvascular pericytes expresses CD105, an MSC marker, whereas malignant cells largely do not. In an in vitro coculture model, SDBMSCs as well as normal human pericytes markedly stimulate the growth of sarcoma cell lines. Conclusions: SDBMSCs/pericytes represent a previously undescribed stromal cell type in sarcoma that may contribute to tumor formation. ©2010 AACR. Less
Purpose: Endosialin/CD248/tumor endothelial marker 1 is expressed in stromal cells, endothelial cells, and pericytes in various tumors; however, few studies have focused ... More
Purpose: Endosialin/CD248/tumor endothelial marker 1 is expressed in stromal cells, endothelial cells, and pericytes in various tumors; however, few studies have focused on expression in malignant cells. Experimental design: We studied expression of endosialin in clinical specimens, cell culture, and animal models and designed an anti-endosialin therapeutic prototype. Results: Fifty human tumor cell lines and 6 normal cell types in culture were assayed by reverse transcription-PCR and/or flow cytometry for endosialin. Cell surface protein was found on 7 sarcoma lines, 1 neuroblastoma, and 4 normal cell types in culture. A fully human anti-endosialin antibody bound to human A-673 Ewing's sarcoma cells and SK-N-AS neuroblastoma cells but not HT-1080 cells. Exposure of cells to an anti-human IgG conjugated to saporin resulted in growth inhibition only of endosialin-expressing cells. Endosialin expression was assessed by immunohistochemistry in 250 clinical specimens of human cancer including 20 cancer subtypes. Endosialin is frequently found in human cancers. Endosialin expression is mainly a perivascular feature in carcinomas, with some expression in stromal cells. In sarcomas, endosialin is expressed by malignant cells, perivascular cells, and stromal cells. Development and characterization of experimental models for studying endosialin biology in sarcomas and evaluating anti-endosialin therapies is presented. Conclusions: Findings suggest that an anti-endosialin immunotoxin might be a promising therapeutic approach for endosialin-positive neoplasia, especially synovial sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and osteosarcoma. Thus, a diagnostic/therapeutic targeted therapeutic approach to treatment of endosialin-expressing tumors may be possible. Less
Expression of bone morphogenetic protein receptor 1A (BMPR1A) is attenuated in the lung vessels of patients with pulmonary arterial hypertension, but the functional impac... More
Expression of bone morphogenetic protein receptor 1A (BMPR1A) is attenuated in the lung vessels of patients with pulmonary arterial hypertension, but the functional impact of this abnormality is unknown. We ablated Bmpr1a in cardiomyocytes and vascular smooth muscle cells (VSMCs) by breeding mice possessing a loxP allele of Bmpr1a (Bmpr1aflox) expressing R26R with SM22alpha-Cre mice. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice died soon after embryonic day 11 (E11) with massive vascular and pericardial hemorrhage and impaired brain development. At E10.5, SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos showed thinning of the myocardium associated with reduced cell proliferation. These embryos also had severe dilatation of the aorta and large vessels with impaired investment of SMCs that was also related to reduced proliferation. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice showed collapsed telencephalon in association with impaired clearing of brain microvessels in areas where reduced apoptosis was observed. Transcript and protein levels of matrix metalloproteinase (MMP) 2 and 9 were reduced in E9.5 and E10.5 SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos, respectively. Knock-down of BMPR1A by RNA interference in human pulmonary artery SMCs reduced MMP2 and MMP9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. RNA interference of MMP2 or MMP9 recapitulated these abnormalities, supporting a functional interaction between BMP signaling and MMP expression. In human brain microvascular pericytes, knock-down of BMPR1A reduced MMP2 activity and knock-down of either BMPR1A or MMP2 caused resistance to apoptosis. Thus, loss of Bmpr1a, by decreasing MMP2 and/or MMP9 activity, can account for vascular dilatation and persistence of brain microvessels, leading to the impaired organogenesis documented in the brain. Less
Expression of bone morphogenetic protein receptor 1A (BMPR1A) is attenuated in the lung vessels of patients with pulmonary arterial hypertension, but the functional impac... More
Expression of bone morphogenetic protein receptor 1A (BMPR1A) is attenuated in the lung vessels of patients with pulmonary arterial hypertension, but the functional impact of this abnormality is unknown. We ablated Bmpr1a in cardiomyocytes and vascular smooth muscle cells (VSMCs) by breeding mice possessing a loxP allele of Bmpr1a (Bmpr1aflox) expressing R26R with SM22alpha-Cre mice. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice died soon after embryonic day 11 (E11) with massive vascular and pericardial hemorrhage and impaired brain development. At E10.5, SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos showed thinning of the myocardium associated with reduced cell proliferation. These embryos also had severe dilatation of the aorta and large vessels with impaired investment of SMCs that was also related to reduced proliferation. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice showed collapsed telencephalon in association with impaired clearing of brain microvessels in areas where reduced apoptosis was observed. Transcript and protein levels of matrix metalloproteinase (MMP) 2 and 9 were reduced in E9.5 and E10.5 SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos, respectively. Knock-down of BMPR1A by RNA interference in human pulmonary artery SMCs reduced MMP2 and MMP9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. RNA interference of MMP2 or MMP9 recapitulated these abnormalities, supporting a functional interaction between BMP signaling and MMP expression. In human brain microvascular pericytes, knock-down of BMPR1A reduced MMP2 activity and knock-down of either BMPR1A or MMP2 caused resistance to apoptosis. Thus, loss of Bmpr1a, by decreasing MMP2 and/or MMP9 activity, can account for vascular dilatation and persistence of brain microvessels, leading to the impaired organogenesis documented in the brain. Less
Endosialin (Tem1) has been identified by two independent experimental approaches as an antigen of tumor-associated endothelial cells, and it has been claimed to be the mo... More
Endosialin (Tem1) has been identified by two independent experimental approaches as an antigen of tumor-associated endothelial cells, and it has been claimed to be the most abundantly expressed tumor endothelial antigen, making it a prime candidate for vascular targeting purposes. Recent experiments have challenged the endothelial expression of endosialin and suggested an expression by activated fibroblasts and pericytes. Thus, clarification of the controversial cellular expression of endosialin is critically important for an understanding of its role during tumor progression and its validation as a potential therapeutic target. We have therefore performed extensive expression profiling analyses of endosialin. The experiments unambiguously demonstrate that endosialin is expressed by tumor-associated myofibroblasts and mural cells and not by endothelial cells. Endosialin expression is barely detectable in normal human tissues with moderate expression only detectable in the stroma of the colon and the prostate. Corresponding cellular experiments confirmed endosialin expression by mesenchymal cells and indicated that it may in fact be a marker of mesenchymal stem cells. Silencing endosialin expression in fibroblasts strongly inhibited migration and proliferation. Collectively, the experiments validate endosialin as a marker of tumor-associated myofibroblasts and tumor vessel-associated mural cells. The data warrant further functional analysis of endosialin during tumor progression and its exploitation as marker of tumor vessel-associated mural cells, expression of which may reflect the non-normalized phenotype of the tumor vasculature. Less
Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels und... More
Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions. As primary cells in culture, pericytes and EPC share many properties such as tube/network formation and response to kinase inhibitors selective for angiogenic pathways. Expression of cell surface proteins including platelet-derived growth factor receptor, vascular cell adhesion molecule, intercellular adhesion molecule, CD105, desmin, and neural growth proteoglycan 2 was similar between pericytes and EPC, whereas expression of P1H12 and lymphocyte function-associated antigen-1 clearly differentiates the cell types. Further distinction was observed in the molecular profiles for expression of angiogenic genes. Pericytes or EPC enhanced the invasion of MDA-MB-231 breast cancer cells in a coculture assay system. The s.c. coinjection of live pericytes or EPC along with MDA-MB-231 cells resulted in an increased rate of tumor growth compared with coinjection of irradiated pericytes or EPC. Microvessel density analysis indicated there was no difference in MDA-MB-231 tumors with or without EPC or pericytes. However, immunohistochemical staining of vasculature suggested that EPC and pericytes may stabilize or normalize vasculature rather than initiate vasculogenesis. In addition, tumors arising from the coinjection of EPC and cancer cells were more likely to develop lymphatic vessels. These results support the notion that pericytes and EPC contribute to malignancy and that these cell types can be useful as cell-based models for tumor vascular development and selection of agents that may provide therapeutic benefit. Less
