Perivascular Network Research Articles

Neuroimaging findings related to glymphatic system alterations in older adults with metabolic syndrome

ObjectiveThe glymphatic system is a glial-based perivascular network that promotes brain metabolic waste clearance. Reduced glymphatic flow has been observed in rat models of type 2 diabetes and hypertension, indicating the role of vascular risk factors in the glymphatic system. However, little is known about how vascular risk factors affect the human glymphatic system. The present study aims to assess the relationships between metabolic syndrome (MetS), a cluster of vascular risk factors, and the glymphatic system function using diffusion magnetic resonance imaging (MRI)-based measures of water diffusivity in the glymphatic compartments, including the brain interstitial space and perivascular spaces around the deep medullary vein. We hypothesized that vascular risk factors are associated with glymphatic dysfunction, leading to cognitive impairment in older adults. MethodsThis cross-sectional study assessed 61 older adults (age range, 65–82 years) who had participated in the Bunkyo Health Study, including 15 healthy controls (mean age, 70.87 ± 4.90 years) and 46 individuals with MetS (mean age, 71.76 ± 4.61 years). Fractional volume of extracellular-free water (FW) and an index of diffusion tensor imaging along the perivascular space (DTI-ALPS) were used as indirect indicators of water diffusivity in the interstitial extracellular and perivenous spaces of white matter, respectively. ResultsAfter adjusting for age, sex, years of education, total Fazekas scale, Pittsburgh sleep quality index (PSQI) score, and intracranial volume (ICV), a significantly (P = 0.030; Cohen's d = 1.01) higher FW was observed in individuals with MetS than in the healthy controls. Furthermore, individuals with MetS had a significantly (P = 0.031; Cohen's d = 0.86) lower ALPS index than the healthy controls, with age, sex, years of education, total Fazekas scale, PSQI score, ICV, fractional anisotropy, and mean diffusivity included as confounding factors. Higher FW was significantly associated with lower ALPS index (r = −0.37; P = 0.004). Multiple linear regression (MLR) with backward elimination analyses showed that higher diastolic blood pressure (BP; standardized β = 0.33, P = 0.005) was independently associated with higher FW, whereas higher fasting plasma glucose levels (standardized β = −0.63, P = 0.002) or higher Brinkman index of cigarette consumption cumulative amount (standardized β = −0.27, P = 0.022) were associated with lower ALPS index. The lower ALPS index (standardized β, 0.28; P = 0.040) was associated with poorer global cognitive performance, which was determined using the Japanese version of the Montreal Cognitive Assessment (MOCA-J) scores. Finally, partial correlation analyses showed a significant correlation between higher FW and lower MOCA-J scores (r = −0.35; P = 0.025) and between higher FW and higher diastolic BP (r = 0.32, P = 0.044). ConclusionThe present study shows the changes in diffusion MRI-based measures reflected by the higher FW and lower ALPS index in older adults with MetS, possibly due to the adverse effect of vascular risk factors on the glymphatic system. Our findings also indicate the associations between the diffusion MRI-based measures and elevated diastolic BP, hyperglycemia, smoking habit, and poorer cognitive performance. However, owing to the limitations of this study, the results should be cautiously interpreted.

Association of MRI Indices of Glymphatic System With Amyloid Deposition and Cognition in Mild Cognitive Impairment and Alzheimer Disease.

The glymphatic system is a whole-brain perivascular network, which promotes CSF/interstitial fluid exchange. Alterations to this system may play a pivotal role in amyloid β (Aβ) accumulation. However, its involvement in Alzheimer disease (AD) pathogenesis is not fully understood. Here, we investigated the changes in noninvasive MRI measurements related to the perivascular network in patients with mild cognitive impairment (MCI) and AD. Additionally, we explored the associations of MRI measures with neuropsychological score, PET standardized uptake value ratio (SUVR), and Aβ deposition. MRI measures, including perivascular space (PVS) volume fraction (PVSVF), fractional volume of free water in white matter (FW-WM), and index of diffusivity along the perivascular space (ALPS index) of patients with MCI, those with AD, and healthy controls from the Alzheimer's Disease Neuroimaging Initiative database were compared. MRI measures were also correlated with the levels of CSF biomarkers, PET SUVR, and cognitive score in the combined subcohort of patients with MCI and AD. Statistical analyses were performed with age, sex, years of education, and APOE status as confounding factors. In total, 36 patients with AD, 44 patients with MCI, and 31 healthy controls were analyzed. Patients with AD had significantly higher total, WM, and basal ganglia PVSVF (Cohen d = 1.15-1.48; p < 0.001) and FW-WM (Cohen d = 0.73; p < 0.05) and a lower ALPS index (Cohen d = 0.63; p < 0.05) than healthy controls. Meanwhile, the MCI group only showed significantly higher total (Cohen d = 0.99; p < 0.05) and WM (Cohen d = 0.91; p < 0.05) PVSVF. Low ALPS index was associated with lower CSF Aβ42 (r s = 0.41, p fdr = 0.026), FDG-PET uptake (r s = 0.54, p fdr < 0.001), and worse multiple cognitive domain deficits. High FW-WM was also associated with lower CSF Aβ42 (r s = -0.47, p fdr = 0.021) and worse cognitive performances. Our study indicates that changes in PVS-related MRI parameters occur in MCI and AD, possibly due to impairment of the glymphatic system. We also report the associations between MRI parameters and Aβ deposition, neuronal change, and cognitive impairment in AD.

Ocular Lymphatic and Glymphatic Systems: Implications for Retinal Health and Disease.

Clearance of ocular fluid and metabolic waste is a critical function of the eye in health and disease. The eye has distinct fluid outflow pathways in both the anterior and posterior segments. Although the anterior outflow pathway is well characterized, little is known about posterior outflow routes. Recent studies suggest that lymphatic and glymphatic systems play an important role in the clearance of fluid and waste products from the posterior segment of the eye. The lymphatic system is a vascular network that runs parallel to the blood circulatory system. It plays an essential role in maintenance of fluid homeostasis and immune surveillance in the body. Recent studies have reported lymphatics in the cornea (under pathological conditions), ciliary body, choroid, and optic nerve meninges. The evidence of lymphatics in optic nerve meninges is, however, limited. An alternative lymphatic system termed the glymphatic system was recently discovered in the rodent eye and brain. This system is a glial cell-based perivascular network responsible for the clearance of interstitial fluid and metabolic waste. In this review, we will discuss our current knowledge of ocular lymphatic and glymphatic systems and their role in retinal degenerative diseases.

Can meditation-based approaches improve the cleansing power of the glymphatic system?

The glymphatic system, first described in 2012, is a brain-wide perivascular network that plays an important role in promoting interstitial metabolic waste removal from the brain. Glymphatic pathway function has been reported to be dramatically diminished in the aging brain. Furthermore, glymphatic system dysfunction has been linked to a spectrum of neurodegenerative diseases including Alzheimer’s disease (AD). This waste clearance pathway of the brain is most active during sleep and is largely disengaged during wakefulness. While norepinephrine (NE) is responsible for suppressing the glymphatic function, electroencephalographic slow-wave (delta) activity has a facilitating effect. An intriguing question is whether these regulators of glymphatic activity can be modulated by meditation-based approaches and whether such approaches have the ability to increase glymphatic function in the awake brain. The present article hypothesizes that meditation-based approaches, such as immersive sound meditation, may have the potential to enhance glymphatic pathway transport and solute clearance by reducing NE and increasing slow-wave activity. If confirmed, meditation could be an attractive approach to promoting healthy brain aging and to preventing neurodegenerative conditions like AD.

Glymphatic System Dysfunction: A Novel Mediator of Sleep Disorders and Headaches.

Sleep contributes to the maintenance of overall health and well-being. There are a growing number of patients who have headache disorders that are significantly affected by poor sleep. This is a paradoxical relationship, whereby sleep deprivation or excess sleep leads to a worsening of headaches, yet sleep onset also alleviates ongoing headache pain. Currently, the mechanism of action remains controversial and poorly understood. The glymphatic system is a newly discovered perivascular network that encompasses the whole brain and is responsible for removing toxic proteins and waste metabolites from the brain as well as replenishing nutrition and energy. Recent studies have suggested that glymphatic dysfunction is a common underlying etiology of sleep disorders and headache pain. This study reviews the current literature on the relationship between the glymphatic system, sleep, and headaches, discusses their roles, and proposes acupuncture as a non-invasive way to focus on the glymphatic function to improve sleep quality and alleviate headache pain.

Geometrically Reduced Modelling of Pulsatile Flow in Perivascular Networks

Flow of cerebrospinal fluid in perivascular spaces is a key mechanism underlying brain transport and clearance. In this paper, we present a mathematical and numerical formalism for reduced models of pulsatile viscous fluid flow in networks of generalized annular cylinders. We apply this framework to study cerebrospinal fluid flow in perivascular spaces induced by pressure differences, cardiac pulse wave-induced vascular wall motion and vasomotion. The reduced models provide approximations of the cross-section average pressure and cross-section flux, both defined over the topologically one-dimensional centerlines of the network geometry. Comparing the full and reduced model predictions, we find that the reduced models capture pulsatile flow characteristics and provide accurate pressure and flux predictions across the range of idealized and image-based scenarios investigated—at a fraction of the computational cost of the corresponding full models. The framework presented thus provides a robust and effective computational approach for large scale in-silico studies of pulsatile perivascular fluid flow and transport.

Measurement of brain clearance and its daytime-dependent efficacy in mice.

Amyloid β (Aβ) plaques are recognized as a main pathological hallmark of Alzheimer´s disease (AD). One important cause for Aβ-plaque formation may be a persistent dysbalance between Aβ production and clearance. In the last years, the so called glymphatic system was identified as main contributor to Aβ clearance from the brain through glia-mediated perivascular networks. Interestingly, the functionality and efficacy of glymphatic clearance is closely related to circadian rhythm integrity. Understanding the correlation between sleep-wake-cycle and its influence on glymphatic clearance will lead to a refinement in future preclinical and clinical AD investigations. To study daytime-dependent changes in brain clearance we established an in vivo protocol to assess the dispersion of a fluorescent dye injected into the hippocampal parenchyma of C57BL6J mice on begin (6 am, dusk for the mice, n = 6) and end (6 pm, dawn for the mice, n = 6) of the light period. Brains were explanted 1 h (n =3 per group) or 3 h (n = 3 per group) post injection and dye dispersion was histologically assessed (Figure 1). First data point to an effect of sleep-wake-cycle on brain clearance, as indicated by elevated fluorescent dye dispersion in the evening. Due to the time delay caused by the Corona pandemic, histologic analyses are still ongoing. However, our results will be ready to be presented at the AAIC 2021. Our data suggest an experimental approach to study day-time dependency of brain clearance in mouse models of AD. We hope that these data will encourage researchers to pay more attention to circadian rhythmicity when studying AD in basic and clinical research.

Dysfunction of the Glymphatic System as a Potential Mechanism of Perioperative Neurocognitive Disorders.

Perioperative neurocognitive disorder (PND) frequently occurs in the elderly as a severe postoperative complication and is characterized by a decline in cognitive function that impairs memory, attention, and other cognitive domains. Currently, the exact pathogenic mechanism of PND is multifaceted and remains unclear. The glymphatic system is a newly discovered glial-dependent perivascular network that subserves a pseudo-lymphatic function in the brain. Recent studies have highlighted the significant role of the glymphatic system in the removal of harmful metabolites in the brain. Dysfunction of the glymphatic system can reduce metabolic waste removal, leading to neuroinflammation and neurological disorders. We speculate that there is a causal relationship between the glymphatic system and symptomatic progression in PND. This paper reviews the current literature on the glymphatic system and some perioperative factors to discuss the role of the glymphatic system in PND.

The Sleeping Brain: Harnessing the Power of the Glymphatic System through Lifestyle Choices

The glymphatic system is a “pseudo-lymphatic” perivascular network distributed throughout the brain, responsible for replenishing as well as cleansing the brain. Glymphatic clearance is the macroscopic process of convective fluid transport in which harmful interstitial metabolic waste products are removed from the brain intima. This paper addresses the glymphatic system, its dysfunction and the major consequences of impaired clearance in order to link neurodegeneration and glymphatic activity with lifestyle choices. Glymphatic clearance can be manipulated by sleep deprivation, cisterna magna puncture, acetazolamide or genetic deletion of AQP4 channels, but how lifestyle choices affect this brain-wide clearance system remains to be resolved. This paper will synthesize existing literature on glymphatic clearance, sleep, Alzheimer’s disease and lifestyle choices, in order to harness the power of this mass transport system, promote healthy brain ageing and possibly prevent neurodegenerative processes. This paper concludes that 1. glymphatic clearance plays a major role in Alzheimer’s pathology; 2. the vast majority of waste clearance occurs during sleep; 3. dementias are associated with sleep disruption, alongside an age-related decline in AQP4 polarization; and 4. lifestyle choices such as sleep position, alcohol intake, exercise, omega-3 consumption, intermittent fasting and chronic stress all modulate glymphatic clearance. Lifestyle choices could therefore alter Alzheimer’s disease risk through improved glymphatic clearance, and could be used as a preventative lifestyle intervention for both healthy brain ageing and Alzheimer’s disease.

Brain Washing System-The System by Which the Wasting Molecule are Removed from Brain

The central nervous system (CNS) is the only organ system of the body which lacks its own waste clearance or lymphatic system, a system which helps in removal of metabolic byproducts and waste solutes. Although the brain plays its role in 25% metabolism of body and comprises only 2% of the total body mass, this high metabolic load needs a proficient system for the removal of waste solutes and for maintaining homeostasis of brain environment. Well-depicted components of waste removal comprise of perivascular fluid flow and phagocytic immune cell functions, nonetheless, the requirement for dynamic clearance of waste from the brain is getting progressively valued. Latest improvements in lymphatic vascular biology confront the recommendation that the brain deficits lymphatic removal system or an equivalent. In this review article, a recently discovered waste removal system, the glymphatic system and its functioning is discussed, keeping in view the experimental studies performed on rodents. The glymphatic system is peri-vascular network dependent of glial cells that serves as a pseudo-lymphatic system in the brain. In the pathway of glymphatic system, cerebrospinal fluid gains entry into brain by means of peri-arterial spaces, moves into the interstitial spaces through perivascular astrocytes and aquaporin-4 channels, and afterwards pushes the peri-venous waste of interstitial fluid (ISF) and its solutes into lymphatic vessels which eventually moves into systemic circulation. This system plays significant role individually and also in combination with authentic lymphatic system in drainage and getting clearance of wastes from the brain.

MRI of Whole Rat Brain Perivascular Network Reveals Role for Ventricles in Brain Waste Clearance

Investigating the mechanisms by which metabolic wastes are cleared from nervous tissue is important for understanding natural function and the pathophysiology of several neurological disorders including Alzheimer’s disease. Recent evidence suggests clearance may be the function of annular spaces around cerebral blood vessels, called perivascular spaces (PVS), through which cerebrospinal fluid (CSF) is transported from the subarachnoid space into brain parenchyma to exchange with interstitial fluid (also known as the glymphatic system). In this work, an MRI-based methodology was developed to reconstruct the PVS network in whole rat brain to better elucidate both PVS uptake and clearance pathways. MR visible tracer (Gd-albumin) was infused in vivo into the CSF-filled lateral ventricle followed by ex vivo high-resolution MR imaging at 17.6 T with an image voxel volume two orders of magnitude smaller than previously reported. Imaged tracer distribution patterns were reconstructed to obtain a more complete brain PVS network. Several PVS connections were repeatedly highlighted across different animals, and new PVS connections between ventricles and different parts of the brain parenchyma were revealed suggesting a possible role for the ventricles as a source or sink for solutes in the brain. In the future, this methodology may be applied to understand changes in the PVS network with disease.

A Low Permeability Microfluidic Blood-Brain Barrier Platform with Direct Contact between Perfusable Vascular Network and Astrocytes

A novel three dimensional blood brain barrier (BBB) platform was developed by independently supplying different types of media to separate cell types within a single device. One channel (vascular channel, VC) is connected to the inner lumen of the vascular network while the other supplies media to the neural cells (neural channel, NC). Compared to co-cultures supplied with only one type of medium (or 1:1 mixture), best barrier properties and viability were obtained with culturing HUVECs with endothelial growth medium (EGM) and neural cells with neurobasal medium supplemented with fetal bovine serum (NBMFBS) independently. The measured vascular network permeability were comparable to reported in vivo values (20 kDa FITC-dextran, 0.45 ± 0.11 × 10−6 cm/s; 70 kDa FITC-dextran, 0.36 ± 0.05 × 10−6 cm/s) and a higher degree of neurovascular interfacing (astrocytic contact with the vascular network, GFAP-CD31 stain overlap) and presence of synapses (stained with synaptophysin). The BBB platform can dependably imitate the perivascular network morphology and synaptic structures characteristic of the NVU. This microfluidic BBB model can find applications in screening pharmaceuticals that target the brain for in neurodegenerative diseases.

The Glymphatic System in Central Nervous System Health and Disease: Past, Present, and Future.

The central nervous system (CNS) is unique in being the only organ system lacking lymphatic vessels to assist in the removal of interstitial metabolic waste products. Recent work has led to the discovery of the glymphatic system, a glial-dependent perivascular network that subserves a pseudolymphatic function in the brain. Within the glymphatic pathway, cerebrospinal fluid (CSF) enters the brain via periarterial spaces, passes into the interstitium via perivascular astrocytic aquaporin-4, and then drives the perivenous drainage of interstitial fluid (ISF) and its solute. Here, we review the role of the glymphatic pathway in CNS physiology, the factors known to regulate glymphatic flow, and the pathologic processes in which a breakdown of glymphatic CSF-ISF exchange has been implicated in disease initiation and progression. Important areas of future research, including manipulation of glymphatic activity aiming to improve waste clearance and therapeutic agent delivery, are also discussed.

The effects of noncoding aquaporin-4 single-nucleotide polymorphisms on cognition and functional progression of Alzheimer's disease

IntroductionThe glymphatic system is a brain-wide perivascular network that facilitates clearance of proteins, including amyloid β, from the brain interstitium through the perivascular exchange of cerebrospinal fluid and interstitial fluid. The astrocytic water channel aquaporin-4 (AQP4) is required for glymphatic system function, and impairment of glymphatic function in the aging brain is associated with altered AQP4 expression and localization. In human cortical tissue, alterations in AQP4 expression and localization are associated with Alzheimer's disease (AD) status and pathology. Although this suggests a potential role for AQP4 in the development or progression of AD, the relationship between of naturally occurring variants in the human AQP4 gene and cognitive function has not yet been evaluated. MethodsUsing data from several longitudinal aging cohorts, we investigated the association between five AQP4 single-nucleotide polymorphisms (SNPs) and the rate of cognitive decline in participants with a diagnosis of AD. ResultsNone of the five SNPs were associated with different rates of AD diagnosis, age of dementia onset in trial subjects. No association between AQP4 SNPs with histological measures of AD pathology, including Braak stage or neuritic plaque density was observed. However, AQP4 SNPs were associated with altered rates of cognitive decline after AD diagnosis, with two SNPS (rs9951307 and rs3875089) associated with slower cognitive decline and two (rs3763040 and rs3763043) associated with more rapid cognitive decline after AD diagnosis. DiscussionThese results provide the first evidence that variations in the AQP4 gene, whose gene product AQP4 is vital for glymphatic pathway function, may modulate the progression of cognitive decline in AD.

Focal Solute Trapping and Global Glymphatic Pathway Impairment in a Murine Model of Multiple Microinfarcts.

Microinfarcts occur commonly in the aging brain as a consequence of diffuse embolic events and are associated with the development of vascular dementia and Alzheimer's disease. However, the manner in which disperse microscopic lesions reduce global cognitive function and increase the risk for Alzheimer's disease is unclear. The glymphatic system, which is a brain-wide perivascular network that supports the recirculation of CSF through the brain parenchyma, facilitates the clearance of interstitial solutes including amyloid β and tau. We investigated whether glymphatic pathway function is impaired in a murine model of multiple microinfarcts induced by intraarterial injection of cholesterol crystals. The analysis showed that multiple microinfarcts markedly impaired global influx of CSF along the glymphatic pathway. Although suppression of global glymphatic function was transient, resolving within 2 weeks of injury, CSF tracers also accumulated within tissue associated with microinfarcts. The effect of diffuse microinfarcts on global glymphatic pathway function was exacerbated in the mice aged 12 months compared with the 2- to 3-month-old mice. These findings indicate that glymphatic function is focally disrupted around microinfarcts and that the aging brain is more vulnerable to this disruption than the young brain. These observations suggest that microlesions may trap proteins and other interstitial solutes within the brain parenchyma, increasing the risk of amyloid plaque formation.SIGNIFICANCE STATEMENT Microinfarcts, small (<1 mm) ischemic lesions, are strongly associated with age-related dementia. However, how these microscopic lesions affect global cognitive function and predispose to Alzheimer's disease is unclear. The glymphatic system is a brain-wide network of channels surrounding brain blood vessels that allows CSF to exchange with interstitial fluid, clearing away cellular wastes such as amyloid β. We observed that, in mice, microinfarcts impaired global glymphatic function and solutes from the CSF became trapped in tissue associated with microinfarcts. These data suggest that small, disperse ischemic lesions can impair glymphatic function across the brain and trapping of solutes in these lesions may promote protein aggregation and neuroinflammation and eventually lead to neurodegeneration, especially in the aging brain.

C-kit positive cells and networks in tooth germs of human midterm fetuses

Numerous studies have attempted to characterize the dental pulp stem cells. However, studies performed on prenatal human tissues have not been performed to evaluate the in situ characterization and topography of progenitor cells. We aimed to perform such a study using of antibodies for CD117/c-kit and multiplex antibody for Ki67+ caspase 3. Antibodies were applied on samples dissected from five human midterm fetuses. Positive CD117/c-kit labeling was found in mesenchymal derived tissues, such as the dental follicle and the dental papilla. The epithelial tissues, that is, dental lamina, enamel organ and oral epithelia, also displayed isolated progenitor cells which were CD117/c-kit positive. Interestingly, CD117/c-kit positive cells of mesenchymal derived tissues extended multiple prolongations building networks; the most consistent of such networks were those of the dental follicle and the perivascular networks of the dental papilla. However, the mantle of the dental papilla was also positive for CD117/c-kit positive stromal networks. The CD117/c-kit cell populations building networks appeared mostly with a Ki67 negative phenotype. The results suggest that CD117/c-kit progenitor cells of the prenatal tooth germ tissues might be involved in intercellular signaling.

Tu1449 3-D Microscopy of the Enteric Glial Network in the Transparent Human Colon Mucosa

Background and Aim: Nestin has been proposed as a marker for neural stem cells and has been also reported to be expressed by enteric neural stem cells (ENSC), along with a variety of other proteins such as p75, α2 integrin, Sox2 and Sox10. However, little is known about the nature and distribution of nestin expressing cells in the gut wall. We hypothesized that nestin expression could be used as a method for isolating and localizing putative ENSC that would enable better molecular characterization of these cells, facilitate their harvesting for therapeutic transplantation efforts as well provide greater insight into their participation, if any, in intrinsic regenerative events. Methods: We studied a transgenic mouse that expressed green fluorescent protein (GFP) under the control of the nestin promoter and using whole tissue confocal microscopy of adult small intestine. Immunohistochemistry with a variety of cell surface markers was used to further subclassify Nestin+ cells in various regions.. Multicolor FACS sorting with a combination of Nestin and cell surface markers was used to sort out different populations of cells, on which In Vitro clonal propagation assays as well as multi-potent differentiation assays were performed. Results: Nestin+ cells form an extensive transmural network in the small intestine, which consists of perivascular cells expressing the surface proteoglycan NG2, as well as others both within and outside of the myenteric ganglia. Within the ganglia, some nestin+ cells express the surface neurotrophin receptor p75, thus identifying them as potential ENSC. When isolated from myenteric plexus and grown in augmented Neurobasal media, we found that only Nestin+ cells propagated in culture and differentiated to produce neurons and glia. In Vitro clonal propagation and differentiation of nestin+ cells sub-sorted for NG2 and p75 showed that only cells expressing both p75 and nestin (and not p75 alone) are capable of proliferating (Mean ± S.E. of percentage proliferative cells in Nestin+p75+ fraction: 1.68 ± 0.52) and differentiating to form neurons and glia In Vitro (Mean ± S.E. of percentage of neurospheres that differentiate to form neurons and glia: 78.26 ± 4.35; only neurons: 0 ± 0; only glia: 2.22 ± 1.96). Conclusions: Nestin and p75 together mark a population of cells that contain ENSC. These cells appear to be localized within the ganglia and are distinct from other nestin+ cells such as those in the perivascular network. Our results suggest that it is possible to obtain enriched populations of ENSC for molecular characterization as well as study their involvement in regenerative events.

Therapeutic Targeting of Lymphoma-Associated Vascular Pericytes,

Abstract 3725Growing evidence has implicated the tumor microenvironment, demarcated by neo-vessels, as an essential disease compartment which influences neoplastic growth and clinical outcome in human lymphoma. The mechanisms whereby stromal cells contribute to the pathogenesis and progression of lymphoma remain poorly understood. Pericytes / vascular smooth muscle cells (VSMC), regulated by platelet-derived growth factor receptor β (PDGFRβ) signaling, play important roles in endothelial cell survival and vascular stability. We hypothesized that the functionality and stability of an effective lymphoma-associated vascular network depends upon the integrity of vascular pericytes, and that biologic agents which selectively target vascular pericytes would induce endothelial dysfunction and impair lymphoma growth. We tested our hypothesis in SCID mice bearing human diffuse large B-cell lymphoma (DLBCL) cell lines by treating with selective PDGFRβ inhibitors including imatinib mesylate and monoclonal anti-PDGFRβ antibody. Treatment with imatinib impaired tumor growth of human lymphoma xenografts including Farage, Karpas422 and OCI-Ly7, while the tumor cells themselves neither expressed PDGFRβ nor were inhibited by imatinib directly. Confocal microscopic analysis of the xenograft tumor tissue showed decreased microvessel density, decreased vascular functional flow, as well as increased vascular leak and apoptosis in the imatinib-treated cohorts, compared to untreated controls. Imatinib targeted tumor-associated PDGFRβ+ pericytes in vivo by inducing apoptosis and disruption of the PDGFRβ+ perivascular network, and PDGFRβ+ VSMC in vitro by inhibition of proliferation. FACS analysis of mononuclear cell suspensions of xenograft tumor tissue revealed decreased numbers of mature pericytes and endothelia, as well as their progenitors, with imatinib treatment. Compared to imatinib, treatment with anti-PDGFRβ monoclonal antibody partially inhibited the growth of Farage lymphomas by depleting pericytes and attenuating angiogenesis. Lastly, microarray analysis of differentially expressed genes in PDGFRβ+ VSMC treated with imatinib showed significant down-regulation of genes implicated in proliferation, survival and angiogenesis, including those within the PI3K/AKT and MAPK/ERK1/2 pathways downstream of PDGFRβ signaling. Taken together, these data suggest that effective targeting of angiogenesis within the tumor microenvironment, without directly targeting tumor cells, can translate into therapeutic effects in DLBCL lymphoma models. PDGFRβ+ pericytes may represent a novel, non-endothelial, anti-angiogenic target for lymphoma therapy. Disclosures:Leonard:glaxosmithkline: Consultancy; EMD Serono: Consultancy; Sanofi Aventis: Consultancy; Pfizer: Consultancy; Immunomedics: Honoraria; Cell Therapeutics: Consultancy; Celgene: Consultancy; Johnson and Johnson: Consultancy; Calistoga: Consultancy; Cephalon: Consultancy; Biogen IDEC: Consultancy; Hospira: Consultancy; Millenium: Consultancy; Novartis: Consultancy; Abbott: Consultancy; Seattle Genetics: Consultancy.

DETECTION OF PERIVASCULAR BLOOD FLOW IN VIVO BY CONTRAST-ENHANCED INTRACORONARY ULTRASONOGRAPHY AND IMAGE ANALYSIS: AN ANIMAL STUDY

1. Acute coronary syndromes are mostly the result of coronary plaque rupture. Diagnostic techniques focusing on the early detection of those plaques that are prone to rupture are still limited. Increased neovascularization in the adventitia and within the atherosclerotic plaque have recently been identified as common features of inflammation and plaque vulnerability. Contrast-enhanced intravascular imaging with microbubbles can be used to trace perfusion. 2. In the present study, we examined the perivascular network of the left anterior descending coronary arteries and left circumflex arteries of four domestic, clinically healthy pigs using intracoronary ultrasound after injection of microbubbles with a differential imaging technique (ACES; Computational Biomedicine Laboratory, University of Houston, Houston, TX, USA). Our aim was to detect blood flow into the coronary lumen and perivascular flow in contrast-enhanced images. Eleven regions of interest (ROI), including perivascular structures, were compared with regard to their grey scale level before and after the injection of SonoVue (0.06 mL/kg; Bracco Diagnostics, Princeton, NJ, USA). 3. A statistically significant (P = 0.018) enhancement was found in the echogenicity of the total perivascular space (adventitial region and perivascular vessels), as indicated by an increase in grey level intensity from 8.33 +/- 0.80 (before) to 10.11 +/- 0.88 (after microbubble injection). A significant enhancement of the 11 selected ROI (perivascular structures) was also recorded after the injection of microbubbles (from 7.92 +/- 2.14 to 14.03 +/- 2.44; P = 0.008). 4. We believe that the detection of perivascular structures with contrast-enhanced intracoronary ultrasonography combined with proper image processing may reinforce our future efforts in the detection of vasa vasorum, an active participant in the creation of acute coronary events.

Bufokinin: actions and distribution in the toad cardiovascular system.

1. Bufokinin is a substance P-like neuropeptide and potent spasmogen isolated from the intestine of the cane toad Bufo marinus. In the present study, we investigated the effects of bufokinin on systemic blood pressure and heart rate in the anaesthetized toad and the distribution of bufokinin-like immunoreactivity in the toad vasculature. 2. Intravenous bufokinin caused a dose-dependent fall in systemic blood pressure (maximum fall 20 mmHg) with an ED50 of 2.9 pmol. At higher doses, the effect was prolonged and blood pressure did not return to baseline within 60 min. There was no significant change in heart rate associated with hypotension. 3. Bufokinin-like immunoreactivity was mapped in whole mounts of toad blood vessels and organs using a mouse polyclonal antibody BK3 (at 1:5000) and the avidin-biotin method. Bufokinin-immunoreactive fibres were associated with most blood vessels examined: a moderately dense perivascular network of varicose fibres was present around renal arteries, with sparser immunoreactive fibres in the ventral aorta, sciatic artery, anterior abdominal vein and hepatic portal vein. 4. Bufokinin-immunoreactive fibres, mainly following blood vessels, were seen in whole mounts of the urinary/bladder and tongue, but not in the air sac. In the heart ventricle, varicose fibres were found in the valve cusps, intracardiac ganglia, epicardium and myocardium close to the endocardium, but not in the rest of the myocardium. 5. The vasodepressor action of bufokinin and the presence of bufokinin-like immunoreactivity in varicose fibres in various vessels suggest a role for bufokinin in haemodynamic regulation and/or sensory nerve function in the toad. The lack of any reflex tachycardia in response to the falls in blood pressure was of note.