Bone development and fracture healing is normal in mice that have a defect in the development of the lymphatic system.

Ectopic lymphatics form in bone and promote bone destruction in diseases such as Gorham-Stout disease, generalized lymphatic anomaly, and kaposiform lymphangiomatosis. However, the role lymphatics serve in normal bone development and repair is poorly understood. The objective of this study was to characterize bone development and fracture healing in mice that have a defect in the development of the lymphatic vasculature. We found that bones in wild-type adult mice and mouse embryos did not have lymphatics. We also found that bone development was normal in Vegfr3 (Chy/Chy) embryos. These mice do not have lymphatics and die shortly after birth. To determine whether lymphatics serve a role in postnatal bone development and fracture healing, we analyzed bones from Vegfr3 (wt/Chy) mice. These mice are viable and have fewer lymphatics than wild-type mice. We found that postnatal bone development and fracture healing was normal in Vegfr3 (wt/Chy) mice. Taken together, our results suggest that lymphatics do not play a major role in normal bone development or repair.

A New Case and Review of Chylothorax in Generalized Lymphatic Anomaly and Gorham-Stout Disease.

Generalized lymphatic anomaly (GLA) and Gorham-Stout disease (GSD) are related diseases involving the lymphatic vasculature. Patients with these diseases frequently develop chylothorax, which can cause respiratory distress, failure, and death. Unfortunately, the optimum treatment for GLA and GSD patients with chylothorax remains unknown. Here we review 64 previously reported cases of chylothorax in GLA and GSD and describe a GLA patient with bilateral chylothorax that was treated with a pleurovenous shunt after multiple other treatments failed. Unfortunately, this shunt was not able to control the patient's effusion, and she succumbed to her disease 3 years after the shunt was placed. Interestingly, our literature review revealed that patients with left-sided effusions had better outcomes than patients with either right-sided or bilateral effusions. Taken together, our report highlights the difficulty in managing chylothorax in patients with GLA or GSD and reveals that a better understanding of the cause of chylothorax is needed so that new therapies can be developed to treat this common complication of GLA and GSD.

Lymphedema-distichiasis syndrome without FOXC2 mutation: evidence for chromosome 16 duplication upstream of FOXC2.

A patient with the classical phenotype of Lymphedema-Distichiasis syndrome (OMIM 153400) is described who showed no mutations in the sequence of FOXC2. Accordingly, a Gene Chip 250k array analysis was undertaken with dense SNP genotyping of the genomic region surrounding the FOXC2 locus on Chromosome 16 followed by copy number evaluation by real time PCR. The latter assay showed evidence of a duplicated region 5' of FOXC2 that could be causative for the patient's striking phenotype, which included both distichiasis and a hyperplastic refluxing lymphatic vascular and lymph node phenotype associated with pubertal onset lymphedema, scoliosis and strabismus.

Novel FOXC2 missense mutation identified in patient with lymphedema-distichiasis syndrome and review.

Lymphedema-distichiasis (OMIM 153400) is a dominantly inherited disorder typically presenting with lymphedema at puberty and distichiasis at birth. The condition has been decisively linked to mutations in the forkhead transcription factor FOXC2 which have been primarily frameshift mutations truncating the protein. We report here a novel missense mutation along with a literature review summarizing reported mutations.

On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death.

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (DeltaPsim) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.

Detection of mitochondrial DNA in living animal cells with fluorescence microscopy.

The detection of mitochondrial DNA (mtDNA) in living human cells could be useful for understanding mitochondrial behaviour during cellular processes and pathological mtDNA depletions. However, until now, human mtDNA has not been visualized in living cells with fluorescence microscopy, although it has been easily detected in organisms with larger mtDNA. Previous reports have stated that mtDNA staining results in homogeneous fluorescence of mitochondria or that animal mitochondria are refractory to DAPI staining. This paper shows that mtDNA of cultured green monkey kidney CV-1 can be stained using a very low concentration of DAPI, then detected by a cooled Photometrics CCD camera with 14-bit resolution detection. Indeed, under these conditions CV-1 cells have small fluorescent spots in the cytoplasm that colocalize with mitochondria, even after mitochondrial movements, uncoupling by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and swelling. These observations have been reproduced for the human fibroblast foreskin cell line HS68. These results and known properties of DAPI as a specific DNA stain strongly suggest that mtDNA can be detected and visualized by fluorescence microscopy in human living cells, with potential developments in the study of mtDNA in normal and pathological situations.

Imaging of cells by autofluorescence: a new tool in the probing of biopharmaceutical effects at the intracellular level.

The success of biopharmaceuticals relies on the ability to have reliable probes to interpret their mechanisms of action in situ at the intracellular level in terms of cell organelles and microcompartments. One of the most effective probes is the endogenous coenzyme NAD(P)H and its fluorescence transients obtained by the microinjection or perfusion of metabolic intermediates and modifiers, in the presence of drugs and inhibitors. The approach in fluorescence microtopography and microspectrofluorimetry is based on the premise that natural cell fluorescence (autofluorescence) holds a decisively greater potential in unravelling intracellular physiopathological processes than extrinsic fluorescence or artificial pseudocolouring. The mounting as a detector of a cooled charge-coupled device camera or alternatively of a non-cooled camera in conjunction with an image intensifier or an investigator (i.e. frame scan accumulator) to enhance sensitivity makes possible the detection of the low-quantum-yield NAD(P)H fluorescence at a level comparable to images previously obtained with high-quantum-yield fluorochromes. The modulation of mitochondrial autofluorescence by rotenone, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and oligomycin, and of cytoplasmic and nuclear autofluorescence by glucose and iodacetamide in CV-1 kidney epithelial cells, Ehrlich-Lettre hypotetraploid CCL77 cells and Saccharomyces cerevisiae, provides examples of the usefulness of fluorescence imaging in the study of biopharmaceuticals. The method goes beyond NAD(P)H to the multiplicity of extrinsic and intrinsic probes already available or in development.

Apoptosis or necrosis following Photofrin photosensitization: influence of the incubation protocol.

Photosensitization using the tumor-localizing porphyrin Photofrin induces cell death both in vitro and in vivo, but the mechanism of cell death is not well understood. Cell lysis (necrosis) and apoptosis have both been observed. The latter seems restricted mainly to lymphoma and epithelial cell lines. To check the influence of the incubation protocol on the cell death mechanism, CV-1 cells were loaded with Photofrin using two different protocols. In both protocols, photosensitized CV-1 cells underwent severe morphological changes before cell death. Many cells treated with protocol 1 (24 h with 1 microgram/mL of Photofrin in culture medium) underwent apoptosis, as demonstrated by plasma membrane blebbing and fragmentation into vesicles, condensation of the chromatin and fragmentation of the nucleus with oligonucleosomic degradation of the DNA. In contrast, cells treated with protocol 2 (1 h with 10 micrograms/mL of Photofrin in phosphate-buffered saline) lysed instead of fragmented, without oligonucleosomic degradation of the DNA. This type of cell death looks much like necrosis. However, early morphological changes suggest that it is, in fact, apoptosis stopped by plasma membrane leakage. It is concluded that apoptosis is primarily induced in CV-1 cells but may be arrested by membrane lysis, depending on the incubation protocol.

Hematoporphyrin derivative (Photofrin) photodynamic action on Ca2+ transport in monkey kidney cells (CV-1).

After 24 h incubation with Photofrin (PF), photodynamic action has been studied on Ca2+ transport in CV-1 cells. A moderate increase of the cytosolic free Ca2+ concentration [Ca2+]i is observed immediately after a dose of irradiation which yields a survival rate of less than 5% at 48 h. In parallel, studies on digitonin-permeabilized cells indicate that such a treatment inhibits endoplasmic reticulum Ca2+ uptake with few alterations of this process in mitochondria. In contrast, ADP-stimulated respiration is impeded and intracellular ATP level decreases. It is suggested that direct damage to endoplasmic reticulum as well as mitochondrial disturbance are the primary mechanisms responsible for a nontransient elevation of [Ca2+]i preceding cell death.

Cytotoxic and photodynamic effects of Photofrin on sensitive and multi-drug-resistant Friend leukaemia cells.

To study cross-resistance to Photofrin (PF) photosensitization, a Friend leukaemia cell line (ADM-RFLC) with a high level of multi-drug resistance (MDR) and the parental sensitive cell line (FLC) have been used. PF uptake measured by HPLC shows a similar intracellular drug accumulation in both cell lines. The ID50s for cell growth inhibition by PF are also similar after exposure in the dark in the two cell lines, while after illumination they are slightly lower in ADM-RFLC than in FLC cells. Moreover, verapamil, known to reverse the MDR phenotype induced by P-glycoprotein over-expression (the drug efflux mechanism), affects equally ADM-RFLC and FLC cells sensitivity to PF. In addition, photodynamic treatment with PF did not reverse the resistance to rhodamine 123 and aclarubicin, but partly reverses resistance of ADM-RFLC cells to antitubulin drugs such as vinblastine or vincristine. These latter results could have clinical application in the treatment of tumours expressing the MDR phenotype.

Structural requirements of simple organic cations for recognition by multidrug-resistant cells.

We previously noted that a wide variety of drugs which are recognized by multidrug-resistant cells (MDR+) are positively charged. However, it remains unclear why and how such a large number of structurally different compounds can be distinguished by MDR+ cells. The majority of the diverse compounds subject to MDR are complex and thereby complicate definitive structure/function characterization of the P-glycoprotein-mediated MDR mechanism. Using a series of simple aromatic (alkypyridiniums) and nonaromatic (alkylguanidiniums) organic cations differing in their lipophilicity by stepwise additions of single alkyl carbons, we demonstrate by growth inhibition studies that a single aromatic moiety and a critical degree of lipophilicity (log P > -1) are required for recognition of these simple organic cations by MDR+ cells. Thus, MDR+ cells are not cross-resistant to the nonaromatic guanidiniums but do show cross-resistance to those aromatic pyridiniums with chain lengths > four. Resistance ratios, as determined by comparison of 50% inhibitory doses in MDR- versus MDR+ cells, increase as a function of increasing chain lengths of these latter simple aromatic compounds. Resistance to pyridinium analogues in MDR+ cells is reversible by co-treatment with nontoxic doses of verapamil. Preliminary uptake data with radioactive analogues further implicate the MDR mechanism of lowered drug accumulation in accounting for resistance to the pyridinium homologues. Utilization of these simple organic cations provides a rational basis for better defining the physical chemical properties of more complex compounds processed by the MDR mechanism and suggests a strategy for designing chemotherapeutic agents with reduced susceptibility to MDR.

Uptake and retention of Photofrin by cultivated human lymphoblastic cells (Reh6): preferential affinity of the cells for a minor component demonstrated by normal phase chromatography.

The uptake of Photofrin by the human cultivated lymphoblastic cell line Reh6 was studied using normal phase high performance liquid chromatography (HPLC) techniques. Relative cellular uptake of eight fractions (uptake/amount of component initially present in the incubation solution) was determined. After 4 h of incubation, protoporphyrin and a small fraction (denoted 4) were incorporated to a greater relative extent than the other fractions. Weakly incorporated components (hematoporphyrin and aggregate-like components) were better retained by cells than the hydrophobic monomeric porphyrins (protoporphyrin and hydroxyethylvinyldeuteroporphyrin). Thus, any benefit gained from a higher uptake was mostly cancelled by a fast release--a situation observed for all fractions except for fraction 4, which displayed both high uptake and good cellular retention. This pattern was not modified when Photofrin concentration or serum percentage was changed. Fraction 4 was further resolved using a gradient system on normal silica. A single component appeared to be mostly responsible for the favorable properties presented by fraction 4, i.e. high uptake and retention within cells. This component was found to correspond to a late eluted peak in the typical reverse-phase HPLC profile of Photofrin. These results emphasize the possible role of minor Photofrin components.

In vitro uptake of dicarboxylic porphyrins by human atheroma. Kinetic and analytical studies.

Human atheromatous aorta segments as well as presumably disease-free control aorta were obtained at autopsy. They were incubated with solutions of various purified dicarboxylic porphyrins including hematoporphyrin (HP) and hydroxyethylvinyldeuteroporphyrin (HVD), and with solutions of Photofrin. Selective labelling of the atheroma was shown by macroscopic and microscopic observations of the characteristic porphyrin fluorescence associated with the atheromatous plaques. The time dependence of the uptake, monitored by absorption spectrophotometry or by high performance liquid chromatography, was inferred from the disappearance of the porphyrins in the incubation medium. Significant binding was observed in the absence of albumin or serum proteins. The uptake of HP was less than that of the more hydrophobic compounds HVD or Photofrin when these porphyrins were used alone. The presence of albumin or serum drastically reduces atheroma labelling. Some competition between HP and HVD for binding sites is also seen. The present results do indicate that hydrophobic porphyrins have an intrinsic affinity for atheroma and that they can be taken up through passive processes. Taking into account previous data on animal models (Photochem. Photobiol. (1989), 731-737), it appears however that, in vivo, interactions with proteins and pharmacokinetics will primarily determine plaque labelling.

Uptake and photodynamic efficiency of hematoporphyrin, hydroxyethylvinyldeuteroporphyrin and hematoporphyrin derivative (Photofrin II): a study with isolated mitochondria.

The uptake of Photofrin II (PFII), hematoporphyrin (Hp) and hydroxyethylvinyldeuteroporphyrin (HVD) by isolated mitochondria was studied using the high performance liquid chromatography (HPLC) technique. The various PFII components show a high affinity for mitochondria. At 5.75 micrograms/ml PFII, their ratio of incorporation was found to be very similar, except for Hp which is about two times less incorporated. These results were reproduced with pure Hp and pure HVD. The uptake of Hp and HVD increases with concentration but, while that of Hp reaches a plateau, the uptake of HVD continues to increase. At a high porphyrin concentration (approximately 10(-5) M), the loss of respiratory control is obtained with the same light dose for Hp and PFII. Taking into account the uptake and the known photophysical parameters of the various porphyrins, the photodynamic efficiency of HVD seems equivalent to that of Hp. The present results and known data on cell photoinactivation suggest that the activity of these porphyrins is mainly dependent on their incorporation.

Singlet molecular oxygen quenching by saturated and unsaturated fatty-acids and by cholesterol.

The rate constants of molecular singlet oxygen quenching by saturated and unsaturated fatty-acids and by cholesterol-membrane critical components - membrane critical components - have been measured by time resolved detection of the 1270 nm phosphorescence of singlet molecular oxygen [O2(1deltag)]. We have determined (i) an increment of 5.7 x 10(2)M(-1)s(-1) per -CH2- in C6D6 and CD3OD for saturated fatty acids between C4 and C20, (ii) an increment of 3 x 10(4)M(-1)s(-1) per non-conjugated cis-double bond for C18 unsaturated fatty acids, identical in C6D6 and DC3OD, (iii) a lower quenching rate constant by a factor of 2.7 for the trans-C16 and trans-C18 as compared to the corresponding cis-monounsaturated fatty acids, (iv) a rate constant of O2x(1deltag) quenching by cholesterol of 5.7 x 10(4)M(-1)s(-1) in benzene. These rate constants are compared to those obtained for other membrane cellular components.

Photofrin II uptake by atheroma in atherosclerotic rabbits. Fluorescence and high performance liquid chromatographic analysis on post-mortem aorta.

Atherosclerotic lesions were induced in normal and Watanabe rabbits by atherogenic diet and stripping of aorta endothelium. The rabbits were injected with Photofrin II and sacrificed two days later. Atheromatous aorta as well as normal aorta from control animals were characterized by their fluorescence spectra using front face excitation. Characteristic emission peaks at 631 and 694 nm were displayed at atheromatous plaques. The excitation spectrum shows a strong band at 394 nm and weaker bands at 446, 504, 536 and 574 nm. Although no fluorescence of normal aorta can be seen by visual inspection, emission with a maximum at 626 nm was detected by spectrofluorimetry. Normal phase high performance liquid chromatography analysis of extracts from atheroma and control aorta were also carried out. The specific labelling of atheroma involves mainly protoporphyrin, hematoporphyrin and also minor components of Photofrin II which are accumulated. Some other components are accumulated but do not appear to be specifically retained by atheroma.

Normal-phase high-performance liquid chromatography of free acid dicarboxylic porphyrins and hematoporphyrin derivative on silica.

The isocratic separation of dicarboxylic porphyrins (hematoporphyrin, hydroxyethylvinyl-deuteroporphyrin and protoporphyrin) and their isomers by normal-phase high-performance liquid chromatography is described. The stationary phase is unmodified silica and the mobile phase consists of acetone-ethyl acetate (1:1, v/v) mixtures containing water and inorganic acids. Retention (capacity factor, k') was found to decrease exponentially with the mole fraction of water (NH2O) and to increase linearly with the concentration of hydrochloric acid, following the relation k' = A [HCl] NH2O-4.85, where A is a constant characteristic of the porphyrin. The effects of the concentration and the nature of the acid used strongly suggest that retention involves a form of the porphyrin in which the inner nitrogens are protonated. The retention is thus partly determined by the basicity of the inner nitrogens, which depends on the electron-donating power of the porphyrin side-chains. Good resolution of the various components of hematoporphyrin derivative was obtained. In comparison with reversed-phase chromatography this method shows a different retention mechanism, appears to yield results of comparable reproducibility and provides complementary information. Possible retention mechanisms based on partition or adsorption equilibria are discussed.

Fundamental aspects in tumor photochemotherapy: interactions of porphyrins with membrane model systems and cells.

Some molecular aspects underlying photochemotherapy and photodiagnosis of tumors with porphyrins are reviewed. The nature of the clinically used photosensitizer HpD is first presented along with structures of molecules found to be efficient in vitro. The possible role of pH in the preferential retention of dicarboxylic porphyrins by tumors is discussed in light of results obtained with membrane models. The uptake of dicarboxylic porphyrins by cells most likely involves passive mechanisms. Cell photoinactivation using a purified porphyrin does not depend upon the incubation time but only on the intracellular concentration of the dye. This likely reflects a poor specificity of the photoinactivation processes with regard to the cellular localization of the dye. The properties which should be presented by more efficient photosensitizers are discussed.