Histone Deacetylase Inhibition and IκB Kinase/Nuclear Factor-κB Blockade Ameliorate Microvascular Proinflammatory Responses Associated With Hemorrhagic Shock/Resuscitation in Mice.

OBJECTIVE: To investigate the consequences of histone deacetylase inhibition by histone deacetylase inhibitor valproic acid and IκB kinase/nuclear factor-κB signaling blockade by IκB kinase inhibitor BAY11-7082 on (microvascular) endothelial cell behavior in vitro as well as in mice subjected to hemorrhagic shock/resuscitation in vivo. DESIGN: Prospective, randomized laboratory investigation using an established mouse model of hemorrhagic shock. SETTING: Research laboratory at university teaching hospital. SUBJECTS: Endothelial cells and C57BL/6 male mice. INTERVENTIONS: Endothelial cells were incubated with tumor necrosis factor-α in the absence or presence of valproic acid or BAY11-7082 in vitro. Mice were subjected to hemorrhagic shock by blood withdrawn until the mean arterial pressure of 30 mm Hg and maintained at this pressure for 90 minutes. At 90 minutes, subgroups of mice were resuscitated with 4% human albumin in the absence or presence of vehicle, valproic acid (300 µg/g body weight) or BAY11-7082 (400 µg per mouse). Mice were killed 1 hour and 4 hours after resuscitation. MEASUREMENTS AND MAIN RESULTS: Valproic acid and BAY11-7082 selectively diminished tumor necrosis factor-α-induced endothelial proinflammatory activation in vitro. In vivo, both systemic and local inflammatory responses were significantly induced by hemorrhagic shock/resuscitation. The decreased histone acetylation in kidneys after hemorrhagic shock/resuscitation was restored by valproic acid treatment. In glomerular endothelial cells, the nuclear translocation of nuclear factor-κB, which was induced by hemorrhagic shock/resuscitation, was eliminated by BAY11-7082 treatment while enhanced in the presence of valproic acid. Both valproic acid and BAY11-7082 significantly attenuated the hemorrhagic shock/resuscitation-induced protein expression of endothelial cell adhesion molecules E-selectin and vascular cell adhesion molecule-1 in the microvasculature of kidneys and liver, although messenger RNA expression levels of these molecules analyzed in whole-organ lysates of kidneys, lungs, and liver were not extensively affected. The reduced protein expression of adhesion molecules was paralleled by diminishing the adhesion/transmigration of neutrophils in kidneys and liver after hemorrhagic shock/resuscitation. CONCLUSION: Suppression of histone deacetylase activity and blockade of IκB kinase/nuclear factor-κB signaling during resuscitation ameliorate microvascular endothelial proinflammatory responses in organs in mice after hemorrhagic shock.

Angiogenesis in synchronous and metachronous colorectal liver metastases: the liver as a permissive soil.

OBJECTIVE: Resection of a primary colorectal carcinoma (CRC) can be accompanied by rapid outgrowth of liver metastases, suggesting a role for angiogenesis. The aim of this study is to investigate whether the presence of a primary CRC is associated with changes in angiogenic status and proliferation/apoptotic rate in synchronous liver metastases and/or adjacent liver parenchyma. METHODS: Gene expression and localization of CD31, HIF-1α, members of the vascular endothelial growth factor (VEGF) and Angiopoietin (Ang) system were studied using qRT-PCR and immunohistochemistry in colorectal liver metastases and nontumorous-adjacent liver parenchyma. Proliferation and apoptotic rate were quantified. Three groups of patients were included: (1) simultaneous resection of synchronous liver metastases and primary tumor (SS-group), (2) resection of synchronous liver metastases 3 to 12 months after resection of the primary tumor [late synchronous (LS-group)], and (3) resection of metachronous metastases >14 months after resection of the primary tumor (M-group). RESULTS: In all 3 groups a higher expression of the angiogenic factors was encountered in adjacent liver parenchyma as compared to the metastases. VEGFR-2 gene expression was abundant in adjacent liver parenchyma in all 3 groups. VEGF-A and VEGFR-1 were prominent in adjacent parenchyma in the SS-group. The SS-group showed the highest Ang-2/Ang-1 ratio both in the metastases and the adjacent liver. This was accompanied by a high turnover of tumor cells. CONCLUSION: In the presence of the primary tumor, the liver parenchyma adjacent to the synchronous liver metastases provides an angiogenic prosperous environment for metastatic tumor growth.

Vascular endothelial growth factor receptor 2 inhibition in-vivo affects tumor vasculature in a tumor type-dependent way and downregulates vascular endothelial growth factor receptor 2 protein without a prominent role for miR-296.

The precise molecular effects that antiangiogenic drugs exert on tumor vasculature remain to be poorly understood. We therefore set out to investigate the molecular and architectural changes that occur in the vasculature of two different tumor types that both respond to vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor therapy. Mice bearing Lewis lung carcinoma (LLC) or B16.F10 melanoma were treated with vandetanib (ZD6474), a VEGFR2/epidermal growth factor receptor (EGFR)/REarranged during Transfection (RET) kinase inhibitor, resulting in a significant 80% reduction in tumor outgrowth. Although in LLC the vascular density was not affected by vandetanib treatment, it was significantly decreased in B16.F10. In LLC, vandetanib treatment induced a shift in vascular gene expression toward stabilization, as demonstrated by upregulation of Tie2 and N-cadherin and downregulation of Ang2 and integrin ß3. In contrast, only eNOS and P-selectin responded to vandetanib treatment in B16.F10 vasculature. Strikingly, vandetanib reduced protein expression of VEGFR2 in both models, whereas mRNA remained unaffected. Analysis of miR-296 expression allowed us to exclude a role for the recently proposed microRNA-296 in VEGFR2 posttranslational control in LLC and B16.F10 in vivo. Our data demonstrate that VEGFR2/EGFR inhibition through vandetanib slows down both LLC and B16.F10 tumor growth. Yet, the underlying molecular changes in the vasculature that orchestrate the antitumor effect differ between tumor types. Importantly, in both models, vandetanib treatment induced loss of its pharmacological target, which was not directly related to miR-296 expression. Validation of our observations in tumor biopsies from VEGFR2 inhibitor-treated patients will be essential to unravel the effects of VEGFR2 inhibitor therapy on tumor vasculature in relation to therapeutic efficacy.

Partial Deletion of Tie2 Affects Microvascular Endothelial Responses to Critical Illness in A Vascular Bed and Organ-Specific Way.

Tyrosine kinase receptor (Tie2) is mainly expressed by endothelial cells. In animal models mimicking critical illness, Tie2 levels in organs are temporarily reduced. Functional consequences of these reduced Tie2 levels on microvascular endothelial behavior are unknown. We investigated the effect of partial deletion of Tie2 on the inflammatory status of endothelial cells in different organs. Newly generated heterozygous Tie2 knockout mice (exon 9 deletion, ΔE9/Tie2) exhibiting 50% reduction in Tie2 mRNA and protein, and wild-type littermate controls (Tie2), were subjected to hemorrhagic shock and resuscitation (HS + R), or challenged with i.p. lipopolysaccharide (LPS). Kidney, liver, lung, heart, brain, and intestine were analyzed for mRNA levels of adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular cell adhesion molecule 1 (ICAM-1), and CD45. Exposure to HS + R did not result in different expression responses of these molecules between organs from Tie2 or Tie2 mice and sham-operated mice. In contrast, the LPS-induced mRNA expression levels of E-selectin, VCAM-1, and ICAM-1, and CD45 in organs were attenuated in Tie2 mice when compared with Tie2 mice in kidney and liver, but not in the other organs studied. Furthermore, reduced expression of E-selectin and VCAM-1 protein, and reduced influx of CD45 cells upon LPS exposure, was visible in a microvascular bed-specific pattern in kidney and liver of Tie2 mice compared with controls. In contrast to the hypothesis that a disbalance in the Ang/Tie2 system leads to increased microvascular inflammation, heterozygous deletion of Tie2 is associated with an organ-restricted, microvascular bed-specific attenuation of endothelial inflammatory response to LPS.

A novel strategy to modify adenovirus tropism and enhance transgene delivery to activated vascular endothelial cells in vitro and in vivo.

To assess the possibilities of retargeting adenovirus to activated endothelial cells, we conjugated bifunctional polyethylene glycol (PEG) onto the adenoviral capsid to inhibit the interaction between viral knob and coxsackie-adenovirus receptor (CAR). Subsequently, we introduced an alphav integrin-specific RGD peptide or E-selectin-specific antibody to the other functional group of the PEG molecule for the retargeting of the adenovirus to activated endothelial cells. In vitro studies showed that this approach resulted in the elimination of transgene transfer into CAR-positive cells, while at the same time specific transgene transfer to activated endothelial cells was achieved. PEGylated, retargeted adenovirus showed longer persistence in the blood circulation with area under plasma concentration-time curve (AUC) values increasing 12-fold compared to unmodified virus. Anti-E-selectin antibody-PEG-adenovirus selectively homed to inflamed skin in mice with a delayed-type hypersensitivity (DTH) inflammation, resulting in local expression of the reporter transgene luciferase. This is the first study showing the benefits of PEGylation on adenovirus behavior upon systemic administration. The approach described here can form the basis for further development of adenoviral gene therapy vectors with improved pharmacokinetics and increased efficiency and specificity of therapeutic gene transfer into endothelial cells in disease.

Endothelial cells internalize and degrade RGD-modified proteins developed for tumor vasculature targeting.

Tumor vasculature can be targeted by peptides containing an RGD (Arg-Gly-Asp) sequence, which bind to alpha(v)beta3 and alpha(v)beta5 integrins on angiogenic endothelial cells. By covalently attaching cyclic RGD-peptides (cRGDfK) to a protein backbone, we prepared a multivalent peptide-protein conjugate with increased affinity for alpha(v)beta3/alpha(v)beta5 integrins. We demonstrated that RGDpep-protein conjugate bound to HUVEC, whereas the conjugate prepared with the control RAD peptide was devoid of any binding. RGDpep-protein conjugate was furthermore functional in inhibiting the adhesion of HUVEC to alpha(v)beta3/alpha(v)beta5 ligand vitronectin, and direct binding of the radiolabeled conjugate to HUVEC was inhibited by alpha(v)beta(3)/alpha(v)beta5-specific RGD peptides. Finally, RGDpep-protein conjugate was shown to be internalized and degraded by HUVEC, a process that could be inhibited by lysosomal degradation inhibitors chloroquine and ammonium chloride. This cellular handling was significantly influenced by the presence of cations, which strongly inhibited internalization. This is the first study that shows direct evidence that primary endothelial cells are capable of internalizing RGD-containing macromolecular proteins. This feature makes them attractive carriers for the intracellular delivery of potent anti-angiogenic drugs into endothelial cells for the treatment of cancer and chronic inflammatory diseases.

Interactions between blood-borne Streptococcus pneumoniae and the blood-brain barrier preceding meningitis.

Streptococcus pneumoniae (the pneumococcus) is a Gram-positive bacterium and the predominant cause of bacterial meningitis. Meningitis is thought to occur as the result of pneumococci crossing the blood-brain barrier to invade the Central Nervous System (CNS); yet little is known about the steps preceding immediate disease development. To study the interactions between pneumococci and the vascular endothelium of the blood-brain barrier prior to meningitis we used an established bacteremia-derived meningitis model in combination with immunofluorescent imaging. Brain tissue of mice infected with S. pneumoniae strain TIGR4, a clinical meningitis isolate, was investigated for the location of the bacteria in relation to the brain vasculature in various compartments. We observed that S. pneumoniae adhered preferentially to the subarachnoid vessels, and subsequently, over time, reached the more internal cerebral areas including the cerebral cortex, septum, and choroid plexus. Interestingly, pneumococci were not detected in the choroid plexus till 8 hours-post infection. In contrast to the lungs, little to no leukocyte recruitment to the brain was observed over time, though Iba-1 and GFAP staining showed that microglia and astrocytes were activated as soon as 1 hour post-infection. Our results imply that i) the local immune system of the brain is activated immediately upon entry of bacteria into the bloodstream and that ii) adhesion to the blood brain barrier is spatiotemporally controlled at different sites throughout the brain. These results provide new information on these two important steps towards the development of pneumococcal meningitis.

Tumor Vascular Morphology Undergoes Dramatic Changes during Outgrowth of B16 Melanoma While Proangiogenic Gene Expression Remains Unchanged.

In established tumors, angiogenic endothelial cells (ECs) coexist next to "quiescent" EC in matured vessels. We hypothesized that angio-gene expression of B16.F10 melanoma would differ depending on the growth stage. Unraveling the spatiotemporal nature thereof is essential for drug regimen design aimed to affect multiple neovascularization stages. We determined the angiogenic phenotype-represented by 52 angio-genes-and vascular morphology of small, intermediate, and large s.c. growing mouse B16.F10 tumors and demonstrated that expression of these genes did not differ between the different growth stages. Yet vascular morphology changed dramatically from small vessels without lumen in small to larger vessels with increased lumen size in intermediate/large tumors. Separate analysis of these vascular morphologies revealed a significant difference in αSMA expression in relation to vessel morphology, while no relation with VEGF, HIF-1α, nor Dll4 expression levels was observed. We conclude that the tumor vasculature remains actively engaged in angiogenesis during B16.F10 melanoma outgrowth and that the major change in tumor vascular morphology does not follow molecular concepts generated in other angiogenesis models.

Site-specific inhibition of glomerulonephritis progression by targeted delivery of dexamethasone to glomerular endothelium.

Glomerulonephritis represents a group of renal diseases with glomerular inflammation as a common pathologic finding. Because of the underlying immunologic character of these disorders, they are frequently treated with glucocorticoids and cytotoxic immunosuppressive agents. Although effective, use of these compounds has limitations as a result of toxicity and systemic side effects. In the current study, we tested the hypothesis that targeted delivery of dexamethasone (dexa) by immunoliposomes to activated glomerular endothelium decreases renal injury but prevents its systemic side effects. E-selectin was chosen as a target molecule based on its disease-specific expression on activated glomerular endothelium in a mouse anti-glomerular basement membrane glomerulonephritis. Site-selective delivery of Ab(Esel) liposome-encapsulated dexamethasone strongly reduced glomerular proinflammatory gene expression without affecting blood glucose levels, a severe side effect of administration of free dexamethasone. Dexa-Ab(Esel) liposomes reduced renal injury as shown by a reduction of blood urea nitrogen levels, decreased glomerular crescent formation, and down-regulation of disease-associated genes. Immunoliposomal drug delivery to glomerular endothelium presents a powerful new strategy for treatment of glomerulonephritis to sustain efficacy and prevent side effects of potent anti-inflammatory drugs.

Preparation and functional evaluation of RGD-modified proteins as alpha(v)beta(3) integrin directed therapeutics.

Tumor blood vessels can be selectively targeted by RGD-peptides that bind to alpha(v)beta(3) integrin on angiogenic endothelial cells. By inhibiting the binding of these integrins to its natural ligands, RGD-peptides can serve as antiangiogenic therapeutics. We have prepared multivalent derivatives of the cyclic RGD-peptide c(RGDfK) by covalent attachment of the peptide to side chain amino groups of a protein. These RGDpep-protein conjugates inhibited alpha(v)beta(3)-mediated endothelial cell adhesion in vitro, while conjugates prepared with a control RAD-peptide showed no activity. Radiobinding and displacement studies with endothelial cells demonstrated an increased affinity of the RGDpep-protein conjugates compared to the free peptide, with IC(50) values ranging from 23 to 0.6 nM, depending on the amount of coupled RGDpep per protein. Compared to the parental RGD-peptide and the related RGD-peptide ligand c(RGDfV), the RGDpep-protein conjugates showed a considerable increase in affinity (IC(50) parent RGDpep: 818 nM; IC(50) c(RGDfV): 158 nM). We conclude that the conjugation of RGD-peptides to a protein, resulting in products that can bind multivalently, is a powerful approach to increase the affinity of peptide ligands for alpha(v)beta(3)/alpha(v)beta(5) integrins.

Targeting of RGD-modified proteins to tumor vasculature: a pharmacokinetic and cellular distribution study.

Angiogenesis-associated integrin alpha(v)beta(3) represents an attractive target for therapeutic intervention because it becomes highly upregulated on angiogenic endothelium and plays an important role in the survival of endothelial cells. Cyclic RGD peptides were prior shown to have a high affinity for alpha(v)beta(3) and can induce apoptosis of endothelial cells. In our laboratory, monocyclic RGD peptides (cRGDfK) were chemically coupled to a protein backbone. Previous results demonstrated that the resulting RGDpep-HuMab conjugate bound with increased avidity to alpha(v)beta(3)/alpha(v)beta(5) on endothelial cells. In our present study, RGDpep-HuMab was injected intravenously and intraperitoneally in B16.F10 tumor-bearing mice to determine its pharmacokinetics and organ distribution. In the tumor, the RGDpep-HuMab conjugate specifically localized at the endothelium as was demonstrated by immunohistochemistry. The control RADpep-HuMab conjugate was not detected in the tumor. Besides tumor localization RGDpep-HuMab was found in liver and spleen associated with macrophages. This uptake by macrophages is probably responsible for the more rapid clearance of RGDpep-HuMab from the circulation than HuMab and RADpep-HuMab. The half-life of RGDpep-HuMab (90 min) was still considerably longer than that of free RGD peptides (<10 min). This prolonged circulation time may be favorable for drug targeting strategies because the target cells are exposed to the conjugate for a longer time period. Taken together these results indicate that RGD-modified proteins are suitable carriers to deliver therapeutic agents into tumor or inflammation induced angiogenic endothelial cells.