Compensatory regrowth of the mouse bladder after partial cystectomy.

Cystectomy is the removal of all or part of the urinary bladder. It has been observed that there is significant regrowth of the bladder after partial cystectomy and this has been proposed to be through regeneration of the organ. Regrowth of tissue in mammals has been proposed to involve compensatory mechanisms that share many characteristics of true regeneration, like the growth of specialized structures such as blood vessels or nerves. However, the overall structure of the normal organ is not achieved. Here we tested if bladder growth after subtotal cystectomy (STC, removal of 50% of the bladder) was compensatory or regenerative. To do this we subjected adult female mouse bladders to STC and assessed regrowth using several established cellular parameters including histological, gene expression, cytokine accumulation and cell proliferation studies. Bladder function was analyzed using cystometry and the voiding stain on paper (VSOP) technique. We found that STC bladders were able to increase their ability to hold urine with the majority of volume restoration occurring within the first two weeks. Regenerating bladders had thinner walls with less mean muscle thickness, and they showed increased collagen deposition at the incision as well as throughout the bladder wall suggesting that fibrosis was occurring. Cell populations differed in their response to injury with urothelial regeneration complete by day 7, but stromal and detrusor muscle still incomplete after 8wks. Cells incorporated EdU when administered at the time of surgery and tracing of EdU positive cells over time indicated that many newborn cells originate at the incision and move mediolaterally. Basal urothelial cells and bladder mesenchymal stem cells but not smooth muscle cells significantly incorporated EdU after STC. Since anti-inflammatory cytokines play a role in regeneration, we analyzed expressed cytokines and found that no anti-inflammatory cytokines were present in the bladder 1wk after STC. Our findings suggest that bladder regrowth after cystectomy is compensatory and functions to increase the volume that the bladder can hold. This finding sets the stage for understanding how the bladder responds to cystectomy and how this can be improved in patients after suffering bladder injury.

Testosterone Modifies Alterations to Detrusor Muscle after Partial Bladder Outlet Obstruction in Juvenile Mice.

Lower urinary tract symptoms secondary to posterior urethral valves (PUV) arise in boys during adolescence. The reasons for this have previously been attributed to increased urine output as boys experience increased growth. Additionally, there are few choices for clinicians to effectively treat these complications. We formed the new hypothesis that increased androgen levels at this time of childhood development could play a role at the cellular level in obstructed bladders. To test this hypothesis, we investigated the role of testosterone on bladder detrusor muscle following injury from partial bladder outlet obstruction (PO) in mice. A PO model was surgically created in juvenile male mice. A group of mice were castrated by bilateral orchiectomy at time of obstruction (CPO). Testosterone cypionate was administered to a group of castrated, obstructed mice (CPOT). Bladder function was assessed by voiding stain on paper (VSOP). Bladders were analyzed at 7 and 28 days by weight and histology. Detrusor collagen to smooth muscle ratio (Col/SM) was calculated using Masson's trichrome stain. All obstructed groups had lower max voided volumes (MVV) than sham mice at 1 day. Hormonally intact mice (PO) continued to have lower MVV at 7 and 28 days while CPO mice improved to sham levels at both time points. In accordance, PO mice had higher bladder-to-body weight ratios than CPO and sham mice demonstrating greater bladder hypertrophy. Histologically, Col/SM was lower in sham and CPO mice. When testosterone was restored in CPOT mice, MVV remained low at 7 and 28 days compared to CPO and bladder-to-body weight ratios were also greater than CPO. Histologic changes were also seen in CPOT mice with higher Col/SM than sham and CPO mice. In conclusion, our findings support a role for testosterone in the fibrotic changes that occur after obstruction in male mice. This suggests that while other changes may occur in adolescent boys that cause complication in boys with PUV, the bladder itself responds to testosterone at the cellular level. This opens the door to a new understanding of pathways that influence bladder fibrosis and could lead to novel approaches to treat boys with PUV.

Binding to and photo-oxidation of cardiolipin by the phthalocyanine photosensitizer Pc 4.

Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Its peroxidation correlates with release of cytochrome c and induction of apoptosis. The phthalocyanine photosensitizer Pc 4 binds preferentially to the mitochondria and endoplasmic reticulum. Earlier Förster resonance energy transfer studies showed colocalization of Pc 4 and cardiolipin, which suggests cardiolipin as a target of photodynamic therapy (PDT) with Pc 4. Using liposomes as membrane models, we find that Pc 4 binds to cardiolipin-containing liposomes similarly to those that do not contain cardiolipin. Pc 4 binding is also studied in MCF-7c3 cells and those whose cardiolipin content was reduced by treatment with palmitate. Decreased levels of cardiolipin are quantified by thin-layer chromatography. The similar level of binding of Pc 4 to cells, irrespective of palmitate treatment, supports the lack of specificity of Pc 4 binding. Thus, factors other than cardiolipin are likely responsible for the preferential localization of Pc 4 in mitochondria. Nonetheless, cardiolipin within liposomes is readily oxidized by Pc 4 and light, yielding apparently mono- and dihydroperoxidized cardiolipin. If similar products result from exposure of cells to Pc 4-PDT, they could be part of the early events leading to apoptosis following Pc 4-PDT.

Structural factors and mechanisms underlying the improved photodynamic cell killing with silicon phthalocyanine photosensitizers directed to lysosomes versus mitochondria.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose-response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

Dynamics and mechanism of bridge-dependent charge separation in pyrenylurea-nitrobenzene pi-stacked protophanes.

Herein are reported the synthesis, structure, and electronic properties of a series of tertiary di- and polyarylureas possessing pyrene and nitrobenzene end groups separated by a variable number of internal phenylenediamine bridging groups. These molecules adopt folded "protophane" structures in which the adjacent arenes are loosely pi-stacked. The behavior of both the pyrene and nitrobenzene singlet states has been investigated by means of femtosecond broadband pump-probe spectroscopy, and the transients have been assigned on the basis of comparison to reference molecules. Femtosecond time resolution permits direct observation of the fast internal conversion process for both the pyrene and nitrobenzene upper singlet states, as well as the intersystem crossing of nitrobenzene. The ultrafast (ca. 100 fs) charge separation of the donor-acceptor urea having no bridging group is attributed to an internal conversion process. The slower charge separation and charge recombination of the donor-acceptor urea having a single bridging group occur via a bridge-mediated superexchange process. Addition of a second bridging unit results in a role reversal for the pyrene singlet state, which now serves as an excited-state acceptor with the bridging units serving as the electron donors. The change in the directionality of electron transfer upon addition of a second bridging phenylenediamine is a consequence of a decrease in the bridge oxidation potential as well as a decrease in the rate constant for single-step superexchange electron transfer.

Convergent synthesis of nonsymmetric pi-stacked protophanes assembled with urea linkers.

A convergent approach has been developed for the preparation of nonsymmetric tertiary arylureas possessing two or three urea linkages. This approach has been used for the preparation of ureas possessing 1-naphthylenyl and 4-nitrophenyl end groups separated by either one or two phenylene diamine bridging units. These ureas were constructed as prototypes for donor-bridge-acceptor systems based on tertiary arylurea architecture. AM1 calculations indicate a preference of these arylureas for folded, protophane structures in which the aryl groups are loosely pi-stacked. Analysis of the (1)H NMR chemical shifts supports the assignment of folded structures in solution. The absorption and luminescence spectra of tertiary ureas possessing 1-naphthylenyl and/or 4-nitrophenyl are reported. The absence of fluorescence and appearance of structured phosphorescence at 77 K are attributed to nitrophenyl-localized lowest energy singlet and triplet states. Localization of excitation on the acceptor chromophore precludes investigation of charge transfer in these systems.

Electronic interactions between pi-stacked chromophores in nonsymmetric tertiary naphthyl di- and polyarylureas.

The synthesis, structure, and spectroscopy of a family of tertiary di- and polyarylureas possessing a naphthyl and several different arene end groups separated by a variable number of internal phenylenediamine linking groups are reported. Molecular modeling and (1)H NMR chemical shift data are consistent with the formation of compact, folded structures in which the arene groups adopt a splayed face-to-face geometry. The structure and solvent dependence of the electronic absorption and emission spectra have been determined and are interpreted with the aid of ZINDO calculations. The electronic absorption spectra are relatively insensitive to the choice of arene end group, the number of linking groups, and the solvent polarity. In contrast, the solution fluorescence is highly dependent upon the structure and solvent polarity. These observations are attributed to a small change in polarity upon excitation of the ground state to a naphthalene-localized Franck-Condon singlet state, which can undergo relaxation to a highly polar emissive state with extensive charge-transfer character.

Electronic interactions in tertiary oligophenylureas.

The syntheses, structures, and spectroscopy of a series of oligomeric tertiary oligophenylureas possessing one to five phenyl rings are reported. A convergent synthetic method employing tertiary monoamine and diamine building blocks is employed. NMR and molecular modeling are indicative of folded structures for all of the oligophenylureas in which adjacent phenyl rings have a splayed face-to-face geometry. NMR chemical shifts, absorption and emission maxima, and electrochemical oxidation potentials are all dependent upon the number of phenyl rings. The addition of a first inner phenyl has a pronounced effect on the chemical shifts, while a second and third inner phenyl have diminished effects. The oxidation potentials of the oligophenylureas display an abrupt decrease upon the addition of the second inner phenyl. The absorption and emission spectra are relatively insensitive to the addition of one to three inner phenyl rings. The electronic structures of the oligophenylureas possessing one to eight rings have been analyzed using ZINDO calculations. The frontier orbitals of the ureas with one to three phenyl rings are localized on a single phenyl ring (the inner ring for the three-ring urea), whereas the frontier orbitals of the higher oligomers are delocalized over two phenyl rings. In all cases, urea-localized n,pi* transitions are lower in energy than the phenyl-localized pi,pi* transitions. The changes in properties with added phenyl rings parallel those previously observed for multilayered cyclophanes; however, they are less pronounced because of weaker coupling between the phenyl rings of the oligophenylureas.