Intact Vasculature Research Articles

Viability and function of the cryopreserved whole rat ovary: comparison between slow-freezing and vitrification

To investigate four different protocols for cryopreservation of the whole rat ovary with intact vasculature to evaluate whether differences exist in post-thawing viability of the ovary after either vitrification or slow freezing. Experimental study. Obstetrics and gynecology department. Immature Sprague-Dawley female rats. Ovaries were isolated with the vascular tree intact up to the bifurcation of the abdominal aorta and were subsequently cannulated. The ovaries were flushed with increasing concentrations of the cryoprotectant dimethyl sulfoxide (DMSO) to either 1.5 or 7 M. The ovaries underwent cryopreservation by vitrification or passive slow freezing. After thawing, the ovaries were subjected to neutral red viability staining to assess the density of viable small follicles and for long-term (48 hours) incubation evaluation of steroid secretion, histology, and apoptosis assay. The follicular viability was decreased in both vitrification groups and in the slow-freezing group with the high concentration of DMSO, as compared with fresh controls. Estradiol levels in the incubation medium followed the same pattern. Light microscopy revealed well-preserved morphology in all groups after 48 hours' incubation. Apoptosis was increased in both vitrified and cryopreserved ovaries. We have developed a new method that can be used in basic studies to improve cryopreservation protocols. Our initial findings suggest that a moderate concentration of the cryoprotectant DMSO is superior to a high DMSO concentration for both vitrification and slow freezing.

Aberrant Kinetics of Bone Marrow–Derived Endothelial Progenitor Cells in the Murine Oxygen-Induced Retinopathy Model

Retinopathy of prematurity (ROP) causes serious blindness because of the vasculopathy that results from the abnormal oxygen dynamics. However, the systemic kinetics of bone marrow-derived endothelial progenitor cells (BM-derived EPCs) during the "postnatal vasculogenesis " of ROP has yet to be elucidated. Thus, the authors investigated the kinetics of BM-derived EPCs using a murine oxygen-induced retinopathy (OIR) model. OIR was induced in C57BL/6J mice by continual aeration with 75% oxygen from postnatal day (P) 7 to P12 that afterward returned to normal room air. The frequency of circulating EPCs (Sca-1(+)/c-Kit(+) cells in blood) in an OIR model estimated by FACS decreased immediately after the hyperoxic phase (P12) and then increased at the hypoxic phase (P17) compared with control. Further, EPC colony-forming assay of BM-Lin(-)/Sca-1(+) (BM-LS) cells exhibited a conversion from the predominant primitive EPC colony production at P12 to the definitive EPC colony at P17. In the OIR retinas of BM-transplanted mice with BM-LS cells of EGFP transgenic mice, there was less incorporation of GFP(+) cells into vascular structures at P12, whereas there was a drastic recruitment into the "tufts " and for the intact vasculature at P17. Moreover, the definitive EPC colony cells intravitreally injected into OIR significantly abrogated pathologic versus primitive vascular growth. Taken together, these findings propose that the deviation of functional bioactivities of BM-derived EPCs contributing to intact vascular development under the abnormal oxygen dynamics may provide important mechanistic insight into pathologic vascular development in ROP.

Initial Binding and Recellularization of Decellularized Mouse Lung Scaffolds with Bone Marrow-Derived Mesenchymal Stromal Cells

Recellularization of whole decellularized lung scaffolds provides a novel approach for generating functional lung tissue ex vivo for subsequent clinical transplantation. To explore the potential utility of stem and progenitor cells in this model, we investigated recellularization of decellularized whole mouse lungs after intratracheal inoculation of bone marrow-derived mesenchymal stromal cells (MSCs). The decellularized lungs maintained structural features of native lungs, including intact vasculature, ability to undergo ventilation, and an extracellular matrix (ECM) scaffold consisting primarily of collagens I and IV, laminin, and fibronectin. However, even in the absence of intact cells or nuclei, a number of cell-associated (non-ECM) proteins were detected using mass spectroscopy, western blots, and immunohistochemistry. MSCs initially homed and engrafted to regions enriched in types I and IV collagen, laminin, and fibronectin, and subsequently proliferated and migrated toward regions enriched in types I and IV collagen and laminin but not provisional matrix (fibronectin). MSCs cultured for up to 1 month in either basal MSC medium or in a small airways growth media (SAGM) localized in both parenchymal and airway regions and demonstrated several different morphologies. However, while MSCs cultured in basal medium increased in number, MSCs cultured in SAGM decreased in number over 1 month. Under both media conditions, the MSCs predominantly expressed genes consistent with mesenchymal and osteoblast phenotype. Despite a transient expression of the lung precursor TTF-1, no other airway or alveolar genes or vascular genes were expressed. These studies highlight the power of whole decellularized lung scaffolds to study functional recellularization with MSCs and other cells.

Near infrared spectroscopy: A diagnostic tool to evaluate effects of radiotherapy in the mandible?

Purpose. This study tests the hypothesis that near infrared spectroscopy can be used to detect changes in haemoglobin oxygenation status in the mandible, and therefore can be used to monitor the deleterious effects of radiotherapy and the possible reversion of these effects with therapeutic ultrasound. Methods. A probe was used to calculate the concentrations of deoxyhaemoglobin in the mandible bone of 30 volunteers with no known malignancies and 10 patients with malignancies in the head and neck region treated with radiotherapy. Results. Although the variability of the measurements was very high, when comparing the right side to the left side of the mandible, the measurements remained relatively similar. There was a great variability between the data for each patient, there was no correlation with age. Conclusions. The near infrared spectroscopy validation for the measurement of deoxyhaemoglobin concentrations in the mandible showed that the variability of the measurements was very high, therefore it is not appropriate to be used diagnostically for the evaluation of radiotherapy effects on the mandibular blood flow and metabolic status. Supply of oxygen is a vitally important function of the local tissue circulation. Along with the nu- trients necessary for life, it is taken up from the cells in the capillary blood and used in the oxidative metabolic process; at the same time, the metabolites are eliminated. Adequate blood flow is required to supply oxygen to the tissue. Physiological blood flow in the tissue requires an anatomically and phys- iologically intact vasculature. If this is impaired as a result of a disease, a state of oxygen deficiency occurs. Depending on its extent, this oxygen deficiency can lead to cell death and tissue necrosis. The timely clinical detection and rectification of a reduced oxygen supply are of crucial importance for the scale of therapy and successful treatment (47).

Vascular rhexis: loss of integrity of coronary vasculature in mice subjected to myocardial infarction

We previously observed gross hemorrhage in plasminogen activator inhibitor type-1 (PAI-1) knockout (PKO) mice with induced myocardial infarction (MI). We hypothesized that it reflected degradation of vessels - a phenomenon we termed vascular rhexis. Accordingly, in the present study we characterized vascular rhexis in C57BL6 mice. MI was induced in 10- to 12-week-old mice by coronary artery ligation for 24, 48, 72 or 96 h. Hemorrhage was quantified by non-cross-reacting enzyme-linked immunosorbent assay of left ventricular (LV) hemoglobin corrected for myoglobin. Degradation of vasculature was quantified by the appearance of alpha smooth muscle actin (alphaSMA) in low salt soluble fractions of LV homogenates (Western blotting) and by immunohistochemistry (residual alphaSMA). Co-staining for CD31 (endothelial cells) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) (a marker of cell death) was used to identify capillary rhexis. PKO mice (n = 9) had marked hemorrhage in infarct zones (432 +/- 27 standard error of mean microL blood/g). Hemorrhage was evident in C57BL6 mice as well (n = 6): 51 +/- 8 microL/g LV 96 h after coronary occlusion compared with 10 +/- 5 microL /g, n = 13 in normal LVs. Residual intact vasculature was reduced 48 h after infarction. Thus, an average of 16 +/- 1.6 small- and medium-sized vessels (n = 5 hearts) were seen compared with 84 +/- 4.8 in normal LVs (n = 3, P < or = 0.05). An approximately three-fold increase in soluble alphaSMA 48 h after MI (2.68 +/- 0.28, n = 6) was seen relative to that in normal LVs defined as 1.0 +/- 0.04, n = 10, P < or = 0.05. Capillary degradation was evident as well, as judged from CD31 and TUNEL co-localization. Vascular rhexis occurs within 48 h after the onset of MI. It may contribute to the early no-reflow phenomenon and to late negative LV remodeling.

Quantitative Assessment of the Vascularity of the Proximal Part of the Humerus

The current consensus in the literature is that the anterolateral branch of the anterior humeral circumflex artery provides the main blood supply to the humeral head. While the artery is disrupted in association with 80% of proximal humeral fractures, resultant osteonecrosis is infrequent. This inconsistency suggests a greater role for the posterior humeral circumflex artery than has been previously described. We hypothesized that the posterior humeral circumflex artery provides a greater percentage of perfusion to the humeral head than the anterior humeral circumflex artery does. In twenty-four fresh-frozen cadaver shoulders (twelve matched pairs), we cannulated the axillary artery proximal to the thoracoacromial branch and ligated the brachial artery in the forearm. In each pair, one shoulder served as a control with intact vasculature and, in the contralateral shoulder, either the anterior humeral circumflex artery or the posterior humeral circumflex artery was ligated. Gadolinium was injected through the cannulated axillary arteries, and magnetic resonance imaging was performed. After imaging, a urethane polymer was injected, and specimens were dissected. For volumetric analysis, the gadolinium uptake on the magnetic resonance imaging was quantified in each quadrant of the humeral head with use of a custom automated program. The gadolinium uptake was compared between the control and ligated sides and between the ligated anterior humeral circumflex artery and ligated posterior humeral circumflex artery groups. The posterior humeral circumflex artery provided 64% of the blood supply to the humeral head overall, whereas the anterior humeral circumflex artery supplied 36%. The posterior humeral circumflex artery also provided significantly more of the blood supply in three of the four quadrants of the humeral head. The finding that the posterior humeral circumflex artery provides 64% of the blood supply to the humeral head provides a possible explanation for the relatively low rates of osteonecrosis seen in association with displaced fractures of the proximal part of the humerus. In addition, protecting the posterior humeral circumflex artery during the surgical approach and fracture fixation may minimize loss of the blood supply to the humeral head.

Enhanced interstitial flow as a contributing factor in neointima formation: (shear) stressing vascular wall cell types other than the endothelium

AS BLOOD FLOWS THROUGH the vascular network, it creates fluid mechanical forces that can be predicted based on established physical principles of fluid dynamics. The major components of flow-generated force in the vasculature include pressure and shear stress, both of which are imparted directly onto the vessel wall. The realization that different patterns (laminar vs. oscillatory), magnitudes (high vs. low), and spatial gradients of shear stress can influence the overall vessel homeostasis has led to an intense investigation into how shear stress regulates the structural and functional phenotype of the blood vessel. Through the work of many dedicated laboratories, we are now certain that shear stress exerts a profound effect on endothelial cells which line all vessels. The endothelium, in turn, can communicate the condition of the hemodynamic environment to which it is exposed to underlying vascular components such as smooth muscle and adventitial fibroblasts, resulting in a propagation of shear stress effects to the entire vessel wall. To illustrate this point, changes in shear stress are perceived by the endothelium and converted in biochemical signals, which are then relayed to enzyme systems that generate vasoactive substances such as nitric oxide (NO). Through a paracrine effect, NO activates the molecular machinery that governs smooth muscle contractility, thereby translating changes in shear stress into an alteration of vessel diameter. In the normal, intact vasculature, the endothelial layer forms an interface between the blood and the remaining components of the vascular wall. Thus vascular smooth muscle and adventitial fibroblasts are remote from the actual hemodynamic shear forces. As a result, the direct influence of shear stress on these mural cell types has received little attention. Rather, the stretch generated via the rhythmic expansion and contraction of the vessel wall with each successive pulse of pressure and bloodflow appears to be the dominant hemodynamic stimuli for smooth muscle and, potentially, adventitial cells. Indeed, cyclic stretch has been shown to activate mechanotransduction pathways that lead to functional responses in these cell, and readers are directed to an excellent review in this area (4). So, if smooth muscle and adventitial cells are shielded, in a relative sense, from shear stress and are most responsive to hemodynamic-imposed stretch, the influences of shear stress would seem to be of little concern in these cell types. However, if one considers the forces produced by the flow of interstitial fluid through the vessel wall, then shear stress forces may in fact be an important factor in regulating smooth muscle cells and vessel wall integrity. This concept may have clinical ramifications given that factors which enhance interstitial flow (i.e., chemical or mechanical injury to the endothelium and inflammation and hypertension induced enhancement of vascular permeability) are associated with vessel remodeling and neointima formation. The basic notion that vascular smooth muscle and fibroblasts are responsive to shear stress has been tested by several investigative teams in the past. Laminar shear stress ranging from 10 to

Decellularization technology application in whole liver reconstruct biological scaffold

To explore an innovative method for preparation of a whole-liver reconstruct scaffold with intact three-dimensional geometry, vasculature and bile duct by decellularization technology. The portal vein was annulated and perfused sequentially with 1%Triton X-100 and 1%SDS for about 4 h, and then was perfused with phosphate buffered saline to dilute SDS residue. The retained structure was evaluated by histological analyses, including macroscopic, Hematoxylin-Eosin staining, Masson's trichrome staining, orcein staining and SEM. The liquid polymer preparation 8% - 10%, which was made of chlorinated poly vinyl chloride (CPVC as solute), acetone (as solvent) and pigment, was injected into portal vein and bile duct to demonstrate the integrity of the portal vein and bile duct. The scaffold was cut into slices with the thickness of about 50 microm and cocultured with C3A cell line. Macroscopic examination showed that the decellularized liver was transparent and intrahepatic Glisson's system could be observed. H&E staining of slices of decellularized liver demonstrated no intact cells or nuclei existed. Masson trichrome staining revealed collagen retained. Orcein staining showed that there were elastic fibers. SEM showed the network of ECM was intact. C3A-to-scaffold co-culture revealed the scaffold of good biocompatibility. Perfusion with detergents through portal vein for liver decellularization was an efficient method to obtain a completely whole-liver scaffold which can be used for hepatic organ reconstruction.

A MEG investigation of somatosensory processing in the rhesus monkey

The use of minimally and non-invasive neuroimaging methods in animal models has sharply increased over the past decade. Such studies have enhanced understanding of the neural basis of the physical signals quantified by these tools, and have addressed an assortment of fundamental and otherwise intractable questions in neurobiology. To date, these studies have almost exclusively utilized positron-emission tomography or variants of magnetic resonance based imaging. These methods provide largely indirect measures of brain activity and are strongly reliant on intact vasculature and normal blood-flow, which is known to be compromised in many clinical conditions. The current study provides the first demonstration of whole-head magnetoencephalography (MEG), a non-invasive and direct measure of neuronal activity, in a rhesus monkey, and in the process supplies the initial data on systems-level dynamics in somatosensory cortices. An adult rhesus monkey underwent three separate studies of tactile stimulation on the pad of the right second or fifth digit as whole-head MEG data were acquired. The neural generators of the primary neuromagnetic components were localized using an equivalent-current-dipole model. Second digit stimulation produced an initial cortical response peaking ∼16 ms after stimulus onset in the contralateral somatosensory cortices, with a later response at ∼96 ms in an overlapping or nearby neural area with a roughly orthogonal orientation. Stimulation of the fifth digit produced similar results, the main exception being a substantially weaker later response. We believe the 16 ms response is likely the monkey homologue of the human M50 response, as both are the earliest cortical response and localize to the contralateral primary somatosensory area. Thus, these data suggest that mechanoreception in nonhuman primates operates substantially faster than that in adult humans. More broadly, these results demonstrate that it is feasible to use current human whole-head MEG instrumentation to record neuromagnetic responses in adult rhesus monkeys. Nonhuman primate models of human disease provide the closest phylogenetic link to humans. The present, non-invasive imaging study could promote exciting translational integration of invasive animal studies and non-invasive human studies, allowing experimentally induced deficits and pharmacological treatments to be interpreted in light of resulting brain network interactions.

Thromboxane A2-induced Bi-directional Regulation of Cerebral Arterial Tone

Myosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces "buffered" vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated.

Quantifying induced electric field strengths during gene transfer to the intact rat vasculature

The application of appropriate electric fields to cells and tissues, termed electroporation, can result in efficient and safe gene transfer. We have shown previously that this approach results in high level gene transfer to and expression in the vasculature of living animals. This paper presents the results of an electric field distribution study of a rat mesenteric vasculature model using the Finite-Element Time-Domain (FETD) technique. A novelty of this work is the use of Debye dispersive dielectric parameters, as the electrical response of biological tissues is inherently frequency-dependent. The results compare fairly well with experimental findings.

When Metabolism Rules Perfusion

See related article, pages 496–505 Since the first description of the AMP-activated protein kinase (AMPK) more than 20 years ago,1 numerous studies have established a pivotal role of this enzyme in the regulation of cellular energy balance. Decreased cellular energy supply (indicated by an increased ATP breakdown and accumulation of AMP) leads to an AMPK-mediated adaptation to allow cellular survival. In doing so, AMPK acts as a cellular “fuel gauge,” such that during nutrient deprivation it will inhibit energy-consuming pathways while it promotes energy producing pathways, eg, fatty acid oxidation or glycolysis. This simple principle is not only operative at the cellular level, because AMPK also governs whole body energy metabolism by the regulation of food intake in the hypothalamus.2 Because the delivery of nutrients and oxygen to the tissue requires an intact vasculature, it is not surprising that AMPK also affects vascular growth and function. Previous studies revealed that AMPK promotes the production of endothelium-derived NO, acts as a proangiogenic signal, inhibits vascular smooth muscle proliferation, and prevents endothelial cell death. All these processes preserve and protect vascular function, an important precondition to provide tissue and cells with nutrients through an intact vasculature to maintain cellular energy levels. Because NO plays a fundamental role in vascular homeostasis,2a the interaction between AMPK and endothelial NO synthase (eNOS) has gathered particular interest. Besides gene expression, the activity of eNOS is largely determined by posttranslational modifications such as multisite phosphorylation, subcellular localization, and eNOS-protein interactions. The eNOS phosphorylation sites associated with enzyme activation include Ser1177 and Ser633, whereas the phosphorylation site at Thr495 inhibits eNOS catalytic activity. Site-specific phosphorylation also occurs at Ser114 and Ser615, but the functional consequences remain unclear. Several upstream kinases which modulate eNOS activity have been identified, including Akt, protein kinase (PK)A, PKC, and …

Smooth Muscle-Specific Gene Delivery in the Vasculature Based on Restriction of DNA Nuclear Import

The two currently employed approaches restricting gene delivery and/or expression to desired cell types in vivo rely on cell surface targeting or cell-specific promoters. We have developed a third approach based on cell-specific nuclear transport of the delivered plasmid DNA. We have previously shown that plasmid nuclear import in non-dividing cells is sequence-specific and have identified a set of cell-specific DNA nuclear targeting sequences that can be used to limit DNA nuclear import to desired cell types. Specifically we have identified elements of the smooth muscle gamma actin (SMGA) promoter that direct plasmid nuclear import selectively in smooth muscle cells (SMCs) in vitro (Vacik et al, 1999, Gene Therapy 6:1006-1014). In the present study, we demonstrate that the SMC-specific DNA nuclear targeting sequence from the SMGA promoter drives nuclear accumulation of plasmids and subsequent gene expression exclusively in the smooth muscle cell layer of the vessel wall in the intact vasculature of rats using electroporation mediated delivery. These results demonstrate that certain DNA nuclear targeting sequences can be used to restrict DNA nuclear import to specific cell types providing a new, novel means of cell targeting for gene therapy.

Contemporary Management of Rectal Cancer

Before the 1980s, rectal cancer was frequently treated with surgery alone, which resulted in high rates of local failure with significant patient morbidity and mortality. Randomized trials performed in the 1980s and 1990s demonstrated that adjuvant chemotherapy decreased rates of local failure and improved survival compared with resection alone. 1,2 These observations led to the adoption of adjuvant radiation therapy and chemotherapy as the standard treatment in the US for patients with resected stage II‐III disease. More recently, proponents of a more thorough and anatomical resection of the mesorectal tissues, i.e. the total mesorectal excision (TME) procedure, reported that local regional failure rates with this approach alone were <10%, questioning the necessity of radiation therapy. To answer this, a large randomized trial was undertaken in The Netherlands comparing neoadjuvant radiation therapy followed by TME with TME only. This trial showed that despite improved surgical techniques, radiation therapy resulted in a significant reduction in local failure (two-year local failure rate 2.4 versus 8.2%; p<0.001). 3 These findings have been supported by the preliminary results of a Medical Research Council (MRC) trial evaluating pre-operative short-course radiotherapy versus selected post-operative combined modality therapy. In this phase III study, 1,350 patients with clinically resectable rectal cancer were randomized to short-course pre-operative radiation therapy (25Gy in 5Gy fractions) and TME versus TME followed by selective post-operative chemoradiation (45Gy in 1.8Gy fractions with 5-fluorouracil (5-FU)) in patients with tumor involvement of the circumferential resection margin. In addition, patients with stage III disease received post-operative adjuvant chemotherapy. In patients undergoing pre-operative radiation therapy, local recurrence was significantly reduced compared with selective post-operative chemoradiation (4.7 and 11.1%, respectively). Furthermore, the three-year disease-free survival rate was significantly improved in patients undergoing pre-operative radiation therapy (79.5 and 74.9%, respectively). 4 These results suggest that even with TME and adjuvant chemotherapy, preoperative radiation improves outcomes over selective post-operative chemoradiation in patients with high-risk disease. Nonetheless, there may be selected stage II or III disease patients who can be treated with TME alone and, therefore, spared chemoradiation, including patients with upper rectal disease with minimal invasion beyond the rectal wall/no nodal involvement (T3N0), as well as disease confined to the rectal wall with minimal nodal involvement (T1‐2, N1). However, given the lack of prospective data supporting this approach, further investigation and validation will be required before general recommendations can be made. Another area of controversy over several decades has centered on the sequencing of radiation therapy relative to surgery. Advocates of preoperative therapy argued that this approach had many benefits, including enhanced rates of sphincter preservation, delivery of therapy to the tumor with an intact vasculature, and improved radiation tolerance. In Germany, a large randomized trial comparing pre-operative with postoperative radiation therapy and chemotherapy confirmed that a preoperative approach resulted in superior treatment compliance, less acute and late toxicity, improved rates of sphincter preservation in patients with low-lying tumors, and a significant improvement in local regional control, again simply by altering the sequence of chemotherapy in relationship to surgery.

Human In vivo Dose-Response to Controlled, Low-Dose Low Linear Energy Transfer Ionizing Radiation Exposure

The effect of low doses of low-linear energy transfer (photon) ionizing radiation (LDIR, <10 cGy) on human tissue when exposure is under normal physiologic conditions is of significant interest to the medical and scientific community in therapeutic and other contexts. Although, to date, there has been no direct assessment of the response of human tissue to LDIR when exposure is under normal physiologic conditions of intact three-dimensional architecture, vasculature, and cell-cell contacts (between epithelial cells and between epithelial and stromal cells). In this article, we present the first data on the response of human tissue exposed in vivo to LDIR with precisely controlled and calibrated doses. We evaluated transcriptomic responses to a single exposure of LDIR in the normal skin of men undergoing therapeutic radiation for prostate cancer (research protocol, Health Insurance Portability and Accountability Act-compliant, Institutional Review Board-approved). Using newly developed biostatistical tools that account for individual splice variants and the expected variability of temporal response between humans even when the outcome is measured at a single time, we show a dose-response pattern in gene expression in a number of pathways and gene groups that are biologically plausible responses to LDIR. Examining genes and pathways identified as radiation-responsive in cell culture models, we found seven gene groups and five pathways that were altered in men in this experiment. These included the Akt/phosphoinositide-3-kinase pathway, the growth factor pathway, the stress/apoptosis pathway, and the pathway initiated by transforming growth factor-beta signaling, whereas gene groups with altered expression included the keratins, the zinc finger proteins and signaling molecules in the mitogen-activated protein kinase gene group. We show that there is considerable individual variability in radiation response that makes the detection of effects difficult, but still feasible when analyzed according to gene group and pathway. These results show for the first time that low doses of radiation have an identifiable biosignature in human tissue, irradiated in vivo with normal intact three-dimensional architecture, vascular supply, and innervation. The genes and pathways show that the tissue (a) does detect the injury, (b) initiates a stress/inflammatory response, (c) undergoes DNA remodeling, as suggested by the significant increase in zinc finger protein gene expression, and (d) initiates a "pro-survival" response. The ability to detect a distinct radiation response pattern following LDIR exposure has important implications for risk assessment in both therapeutic and national defense contexts.

Serotonin Signaling in Pulmonary Hypertension

See related article, pages 1323–1330 Serotonin (5-HT, 5-hydroxytryptamine) has long been recognized as one of the most potent naturally occurring pulmonary vasoconstrictors.1 It was first implicated in the pathogenesis of pulmonary arterial hypertension (PAH) after an outbreak of the disease in Switzerland in the 1960’s among patients taking aminorex fumarate, an appetite suppressant that inhibits serotonin uptake by platelets.2 Since that time further outbreaks of PAH have been identified in Europe and the USA associated with the use of fenfluramine-derivate anorexigens,3–5 eventually leading to their withdrawal from the world market in 1997. Although this was, at least in retrospect, a predictable tragedy, it has ironically opened avenues of research into the biology of serotonin signaling in PAH. As fenfluramine-derivatives are substrates for the serotonin transporter (5-HTT, SERT) proteins,6 this suggests that abnormal SERT expression or functional activity could play a role in the pathogenesis of PAH. There is now a body of evidence supporting this hypothesis that provides hope for the development of effective therapeutic strategies targeting specific components of this signaling pathway in patients with these diseases. Most of the serotonin produced in the body is secreted by enterochromaffin cells of the intestine into the portal circulation where it is partially metabolized by the liver. However, levels of free circulating serotonin are maintained in the low nanomolar range through energy-dependent SERT-mediated transport into platelets. This led some researchers to hypothesize that fenfluramines might cause PAH by increasing free plasma levels of serotonin. However, this hypothesis is inconsistent with the observation that chronic treatment with fenfluramine-derivatives if anything reduces plasma levels of serotonin.6 This suggests that other SERT-related effects promote PAH in susceptible patients. This is supported by the observation that patients with idiopathic PAH have increased frequency of the so called L-type polymorphism …

Blood Pressure Reduction and Tissue-Type Plasminogen Activator Release

Hypertension is associated with increased risk of thrombotic events including myocardial infarction and stroke. The endothelium plays an important role in limiting intravascular thrombosis, inhibiting coagulation and platelet aggregation, and promoting fibrinolysis. Hrafnkelsdottir et al1 have previously described impaired vascular tissue-type plasminogen activator (t-PA) release in individuals with essential hypertension. In the current issue of Hypertension ,2 this group reports that treatment with either an angiotensin-converting enzyme (ACE) inhibitor or a calcium channel blocker increases stimulated t-PA release from the forearm vasculature. t-PA is synthesized and stored in small, dense granules in the vascular endothelium and released in response to such stimuli as Factor Xa, thrombin, bradykinin, and catecholamines, as well as substance P, the vasopressin analogue desmopressin, methacholine, tumor necrosis factor α, and adenosine and uridine triphosphates (Figure).3 Although some studies suggest that t-PA colocalizes with von Willebrand factor in Weibel-Palade bodies, the preponderance of evidence suggests that t-PA is stored in distinct granules and that the release of t-PA and von Willebrand factor are differentially regulated.4 Although shear stress induces t-PA release from cultured endothelial cells, t-PA release from the intact vasculature appears to …

Mechanisms of Endothelial Cell Heterogeneity in Health and Disease

See related article, pages 200–208 The endothelium is an expansive spatially distributed organ.1 Endothelial cells participate in a large number of physiological processes including the control of vasomotor tone, the trafficking of cells and nutrients, the regulation of permeability, and the maintenance of blood fluidity. In addition, the endothelium mediates new blood vessel formation, contributes to the balance of pro- and antiinflammatory mediators, and may play a role in antigen presentation. In accomplishing these tasks, the endothelium exhibits a remarkable “division of labor”. For example, arteriolar endothelium is primarily responsible for mediating vasomotor tone; endothelium in postcapillary venules regulates leukocyte trafficking; capillary endothelial cells display organ-specific barrier properties (eg, blood brain barrier versus fenestrated, discontinuous endothelium in hepatic sinusoids); and endothelial cells from different vascular beds balance local hemostasis via the expression of site-specific patterns of anticoagulants and procoagulants.2 In recent years, in vivo phage display and direct proteome mapping of the intact vasculature have revealed a rich diversity in endothelial cell surface markers.3,4 Any consideration of the mechanisms underlying endothelial heterogeneity is best framed around the time-honored debate of nature versus nurture (which will be addressed here in reverse order) (Figure). Mechanisms of endothelial cell heterogeneity. Relative importance of epigenetics and microenvironment in mediating site-specific phenotypes is indicated by +. The table is designed to provide a conceptual framework; the scores are largely speculative and will require ongoing experimental validation. ### Nurture Site-specific endothelial cell phenotypes may be initiated and maintained by signals residing in the extracellular environment. The endothelium is analogous to a barcode reader, constantly taking inventory of its surrounding extracellular environment on the luminal side (circulating blood and its constituents), the abluminal side, and at the endothelial junctions. Environmental cues may be classified into biomechanical or biochemical. Biochemical forces include shear stress and strain. …

Notch Signaling Represses Myocardin-induced Smooth Muscle Cell Differentiation

Notch signaling is essential for vascular patterning and response of the vasculature to injury and growth factor stimulation. Despite these findings, the molecular basis of Notch signaling in the vasculature is poorly understood. Here we report that activation of Notch signaling mediated through members of the HRT family of basic helix-loop-helix transcription factors represses smooth muscle cell (SMC) differentiation and expression of genes encoding smooth muscle cell contractile markers. Activation of Notch receptors by Jagged1 or forced expression of the constitutively active Notch1 intracellular domain in C3H10T1/2 fibroblasts inhibited myocardin-dependent transcription of SMC-restricted genes and activity of multiple SMC-restricted transcriptional regulatory elements. Consistent with these findings, forced expression of HRT2 inhibited myocardin-induced expression of SMC-restricted genes and activity of SMC-restricted transcriptional regulatory elements. Moreover, forced expression of HRT2 repressed transcription of multiple SMC-restricted transcriptional regulatory elements in A10 SMCs. The repressive function of HRT2 was not mediated via the capacity of HRT2 to bind SMC CArG elements or by disruption of myocardin-SRF protein complexes. Structure-function analyses of HRT2 indicated that repression required the basic DNA binding domain and additional C-terminal sequence. Taken together, these results demonstrate that Notch signaling represses myocardin-dependent SMC transcription. These data are consistent with a model wherein Notch signaling represses SMC differentiation and maintenance of the contractile SMC phenotype.

Medial calcinosis of monckeberg limiting fibula flap reconstruction of the head and neck.

Objectives: To review medial calcinosis of Monckeberg and how this process can limit use of the fibula free flap for head and neck reconstruction. Methods: A 82-year-old man presented with advanced osteoradionecrosis of the left hemimandible after undergoing curative radiation therapy for an advanced oral cavity carcinoma. He was free of recurrent disease and had failed standard therapy and hyperbaric oxygen therapy. Although preoperative MRA demonstrated intact lower extremity vasculature, at the time of surgery rigid calcification was noted in the peroneal and tibial vessels. Successful revacularization was completed, but ultimately flap failure occurred. Debridement and local flap closure was required. Results: Pathologic evaluation revealed extensive medial calcinosis of Monckeberg involving the peroneal artery. Subsequent plain films of lower extremities demonstrated arterial calcinosis in the setting of the normal flow study (MRA). Conclusion: Pathologically, medial calcinosis of Monckeberg is limited to the medial layer of small and medium sized arteries. Distinct from the intimal calcification noted with atherosclerosis (notable on MRA), Monckeberg’s calcification does not significantly affect the vessel lumen size or flow. Yet the vessel rigidity can limit microvascular revascularization as well and pedicle geometry. Although flow studies such as MRA and standard angiography may not demonstrate the process, Monckeberg calicification is obvious on plain films of the lower extremity and these are recommended in patients with risk factors such as long-standing diabetes mellitus. Alternative reconstructive methods should be considered in such cases or if this rigid vessel calcification is noted on flap harvest.