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. 2020 Feb 25;10(1):57–65.

Assessing bladder hyper-permeability biomarkers in vivo using molecularly-targeted MRI

Rheal A Towner 1,2, Nataliya Smith 1, Debra Saunders 1, Megan Lerner 3, Beverley Greenwood-Van Meerveld 4, Robert E Hurst 5
PMCID: PMC7076299  PMID: 32211219

Abstract

The objective was to investigate if some of the key molecular players associated with bladder hyper-permeability in interstitial cystitis/bladder pain syndrome (IC/BPS) could be visualized with molecularly-targeted magnetic resonance imaging (mt-MRI) in vivo. IC/BPS is a chronic, painful condition of the bladder that affects primarily women. It has been demonstrated over the past several decades that permeability plays a substantial role in IC/BPS. There are several key molecular markers that have been associated with permeability, including glycolsaminoglycan (GAG), biglycan, chondroitin sulfate, decorin, E-cadherin, keratin 20, uroplakin, vascular endothelial growth factor receptor 1 (VEGF-R1), claudin-2 and zonula occludens-1 (ZO-1). We used in vivo molecularly-targeted MRI (mt-MRI) to assess specific urothelial biomarkers (decorin, VEGF-R1, and claudin-2) associated with bladder hyper-permeability in a protamine sulfate (PS)-induced rat model. The mt-MRI probes consisted of an antibody against either VEGF-R1, decorin or claudin-2 conjugated to albumin that had also Gd-DTPA (gadolinium diethylene triamine penta acetic acid) and biotin attached. mt-MRI- and histologically-detectable levels of decorin and VEGF-R1 were both found to decrease following PS-induced bladder urothelial hyper-permeability, whereas claudin-2, was found to increase in the rat PS model. Verification of the presence of the mt-MRI probes were done by targeting the biotin moiety for each respective probe with streptavidin-hose radish peroxidase (HRP). Levels of protein expression for VEGF-R1, decorin and claudin-2 were confirmed with immunohistochemistry. In vivo molecularly-targeted MRI (mt-MRI) was found to successfully detect alterations in the expression of decorin, VEGFR1 and claudin-2 in a PS-induced rat bladder permeability model. This in vivo molecularly-targeted imaging approach has the potential to provide invaluable information to enhance our understanding of bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing novel therapeutic strategies.

Keywords: Claudin-2, vascular endothelial growth factor receptor 1 (VEGF-R1), decorin, molecularly-targeted magnetic resonance imaging (mt-MRI), interstitial cystitis (IC), in vivo, protamine sulfate (PS), rats, intravesical

Introduction

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects up to 7% of females in the U.S.A. [1], and is associated with a significant minority (>30%) of female patients with chronic pelvic pain [2]. The prevalence of IC/BPS is markedly higher (100-300/100,000 women) than previously thought [3]. Frequency, urgency and pain during bladder filling are the most commonly reported symptoms of IC/BPS [4]. IC/BPS is thought to be either a type of hypersensitivity disorder that affects bladder and other somatic/visceral organs with many overlapping symptoms and pathophysiology, or a continuum of painful vs. non-painful overactive bladder syndrome [5].

Not only is the etiology of IC/BPS unknown, but there is considerable debate as to whether it occurs due to events within or from outside the bladder, or whether both occur. What is currently known is that permeability does play a role. Parsons has long argued that a key etiologic factor involves permeability that is produced by loss of the glycosaminoglycan (GAG) layer that he argues is a major component of the bladder defenses that produce impermeability [6-15]. Parsons demonstrated increased uptake of instilled urea from the bladders of IC patients [16], and Buffington showed altered excretion of injected fluorescein, presumably representing recycling of the dye in the urine [17]. We also recently used contrast-enhanced MRI to directly measure bladder permeability in IC patients [18]. Increased permeability was thought to result in pain from the same mechanism as produces pain in bladder cancer, CIS, and infection, namely loss of the specialized “umbrella” cells that form the luminal layer of cells and are responsible for maintaining the impermeability barrier. Impermeability is maintained by multiple “defense molecules” [19], tight junctions [20] and the uroplakin plaques [21], and also the GAG layer [22].

Uroplakin [19,23-25], keratin 20 [23,26], chondroitin sulfate [23,27-29], decorin [23,30], VEGF-R1 [31,32], and claudin-2 [33] are all known bladder urothelium markers. In feline interstitial cystitis, which is thought to emulate human IC/BPS, there were definite changes in luminal GAG observed, as well as several altered protein expressions [23]. Abnormalities in protein expression included biglycan, chondroitin sulfate, decorin, E-cadherin, keratin, uroplakin and ZO-1 [23]. In particular, chondroitin sulfate, biglycan and decorin levels were substantially low in expression [23].

Intravesical administration of Gd-DTPA in conjunction with the use of dynamic CE-MRI (DCE-MRI) was developed by our group and validated in a rat pre-clinical model as well as in a small cohort of IC patients. We developed an in vivo MRI test to assess increased bladder urothelial permeability in a pre-clinical rat model following protamine sulfate (PS) exposure using a dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) approach [34]. This method involves intravesical administration of a contrast agent, Gd-DTPA, to monitor leakage or uptake of this agent through the bladder wall. The enhanced contrast MR imaging approach was found to detect bladder urothelium leakage of the contrast agent in rat bladder urothelial [34]. The CE-MRI approach can also be used to assess the effect of therapeutic intervention regarding decreased bladder urothelium permeability. The advantage of the DCE-MRI approach is that 2D and 3D regions of the bladder urothelium can be assessed to establish if there are areas of the bladder wall that are more susceptible to bladder permeability alterations. Gd-DTPA MRI contrast agent was also taken up and significantly retained in human IC bladder urothelium, compared to normal control bladders [18], and that this method could potentially be used as a diagnostic tool to help evaluate bladder hyper-permeability alterations in IC patients. The agent was well tolerated by all IC patients tested, indicating this technology could be translated rapidly to the clinic.

We wanted to investigate if some of the key molecular players associated with bladder hyperpermeability could be visualized with molecularly-targeted MRI. In this study, we used in vivo molecular-targeted MRI to assess specific urothelial biomarkers (decorin, VEGF-R1, and claudin-2) associated with bladder permeability alterations in a protamine sulfate (PS)-induced rat model for bladder hyper-permeability.

Materials and methods

Animals

All experiments involving animals was approved by the Oklahoma Medical Research Foundation (OMRF) Institutional Animal Care and Use Committee (IACUC), and followed the National Research Council guide for the care and use of laboratory animals. Female rats (ovarectimized (OVX)) (200-250 g) were anesthetized with isoflurane (1.5-3.0% with 800-1,000 mL O2) for MRI experiments. PS was administered at a concentration of 1 mg/mL (in saline) in a total volume of 800 µL via an intravesical catheter. A lubricated sterile catheter (18 gauge) was used to transurethally catheterize each animal. PS was instilled in the bladder for 10 min. PS was then removed from the bladder using abdominal pressure and followed by 3 saline flushes (400 µL per flush). Bladder permeability assessments were made at 24 hr following exposure to PS (n=4 for each mt-MRI group). Sham controls (n=4) were administered saline (800 µL) instead of PS.

Molecular-targeted MR imaging probes

Specific probes were synthesized by coupling antibodies against each biomarker to albumin in the Gd-DTPA-albumin-biotin construct. We have previously used this approach for molecularly-targeted MRI probes for in vivo VEGF-R2 [35,36], iNOS [37] and free radical [38-43] detection. The macromolecular contrast material, biotin-BSA-Gd-DTPA, was prepared using a modification of the method of Dafni et al. [44]. The biotin moiety in the contrast material was added to allow histological localization. Biotin-BSA-Gd-DTPA was synthesized as described in Coutinho de Souza et al. [40]. A solution of biotin-BSA-Gd-DTPA was added directly to the solution of antibody (anti-DMPO, 200 μg/mL) for conjugation through a sulfo-NHS (N-succinimidyl-S-acetylthioacetate)-EDC (N-succinimidyl 3-(2-pyridyldithio)-propionate) link between albumin and antibody according to the protocol of Hermanson [45]. Sulfo-NHS was added to the solution of biotin-BSA-Gd-DTPA and EDC. This activated solution was added directly to the antibody (anti-DMPO, 200 μg/mL) for conjugation. The mixture was left to react for at least 2 h at 25°C in the dark. The product was lyophilized and subsequently stored at 4°C and reconstituted to the desired concentration for injections in phosphate buffer saline (PBS). Each probe (0.1 mg/kg bw; 200 µL diluted in saline for 800 µl total volume) was administered via an intravesical catheter, instilled for an hour, and flushed with saline. For decorin-targeted, VEGF-R1-targeted or claudin-2-targeted molecular MRI, rats (PS-treated, or their respective shams) were administered either a biotin-Gd-DTPA-albumin-anti-decorin (anti-decorin) probe, a biotin-Gd-DTPA-albumin-anti-VEGF-R1 (anti-VEGF-R1 probe), or a biotin-Gd-DTPA-albumin-anti-claudin-2 (anti-claudin-2 probe) (i.v.; 1 mg/kg in 800 µL, and scanned at pre-contrast, and 4 hours post-targeted contrast agents (anti-decorin, anti-VEGF-R1, or anti-claudin-2 probes).

Magnetic resonance imaging (MRI)

MRI experiments were conducted on a 7 Tesla 30 cm-bore Bruker Biospec MRI system. A RARE (Rapid acquisition with relaxation enhancement) variable TR sequence was used to detect changes in MRI signal intensities and T1 relaxation maps using a TR (repetition time) =200, 400, 800, 1200 and 1600 ms, TE (echo time) =15 ms, 20 transverse 1 mm-thick slices, a field of view of 3.5 × 3.5 cm2, matrix 256 × 256, with an in-plane resolution of 137 × 137 µm2 (as previously done [35-43]) resulting from the administration of the molecular-targeted MRI probes. Pre- and post-probe administration images were taken to establish specific uptake of each probe in the bladder urothelium.

MRI analysis

MRI signal intensities and T1 values were measured from regions-of-interest (ROIs) within images (ROIs were taken in bladder walls from images and T1 maps, along with corresponding regions in sham animal datasets, as displayed on Paravision (v 5.0, Bruker Biospin)).

Histology and immunohistochemistry (IHC)

Ex vivo bladder urothelium tissue samples, utilizing MRI coordinates from the MR images, were assessed by histology and immunohistochemistry (IHC) to characterize which bladder urothelium markers were altered. Quantification of IHC markers was done using an Aperio ScanScope Image Analysis System [46]. The bladders of each animal were removed, preserved in 10% neutral buffered formalin, and processed routinely. Hematoxylin-eosin staining: tissues were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Sections were deparaffinized, rehydrated, and stained according to standard protocols. Several reagents were produced by Vector Labs Inc. (VLI) in Burlingame, CA. Histological sections (5 µm) embedded in paraffin and mounted on HistoBond® Plus slides (Statlab Medical Products, Lewisville, TX) were rehydrated and washed in Phosphate Buffered Saline (PBS). The sections were processed using the ImmPRESS™ VR Reagent Anti-Rabbit IgG Peroxidase (VLI cat #MP-6401). Antigen retrieval (pH 6 citrate antigen unmasking solution; (VLI cat#H-3300) was accomplished via 20-minutes in a steamer followed by 30-minutes cooling at room temperature. Sections were treated with a peroxidase blocking reagent (Bloxall, VLI cat#SP-6000), followed by 2.5% normal horse serum to inhibit nonspecific binding. Anti-claudin-2, anti-VEGFR1, or anti-decorin antibodies (Invitrogen, ThermoFisher Scientific, Waltham, MA) were separately applied to each section, from bladders not undergoing mt-MRI (n=5/group × 2 groups; saline vs. PS), and following incubation overnight (4°C) in a humidified chamber, sections were washed in PBS, the ImmPRESS VR reagent was applied according to the manufacturer’s directions. Sections for streptavidin horse radish peroxidase (SA-HRP), for the bladders that underwent mt-MRI (n=5/group × 2 groups; saline vs. LPS), where the streptavidin targets the biotin moiety of the mt-MRI probes, were processed as above, except they were incubated overnight with ready to use (RTU) Strp-HRP (VLI cat#SA-5704). Appropriate washes were in PBS. Slides were incubated with NovaRed® (VLI cat#SK-4805) chromogen for visualization. Counterstaining was carried out with Hematoxylin QS Nuclear Counterstain (VLI). Appropriate positive and negative tissue controls were used.

Statistical analysis

Levels of the molecular-targeted MRI imaging probes were compared between PS- or saline-treated rats, using bivariate correlation coefficients. Data analyses used IBM SPSS Statistics (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp). P-values <0.05 were considered significant.

Results

Figure 1 is an illustration of the molecularly-targeted probes used to assess levels of claudin-2, VEGF-R1 and decorin. Figures 2, 3 and 4 indicates the feasibility of conducting in vivo molecular-targeted MR imaging for bladder permeability biomarkers claudin-2 (Figure 2), VEGF-R1 (Figure 3) and decorin (Figure 4), in PS-exposed rat bladders, respectively. Claudin-2 levels are significantly increased (P<0.05) in PS-treated rat bladders (Figure 2A, 2C), compared to saline controls (n=4/group), which had low levels (Figure 2B, 2C). Streptavidin-horse radish peroxidase (SA-HRP) was used to target the anti-claudin-2 probe in PS-(Figure 2D) and saline-(Figure 2E)-treated rat bladders. Note the higher detection of the anti-claudin-2 probe in the PS-treated rat bladder, compared to the saline-treated bladder. Conversely, VEGF-R1 and decorin levels were significantly lower in PS-treated rat bladders (n=4/group; P<0.01 and P<0.05, respectively) compared to controls (n=4 for both; Figures 3 and 4, respectively). Note low levels of both VEGF-R1 and decorin in the bladder dome regions (Figures 3Bii and 4Bii, respectively). SA-HRP was used to target the anti-VEGF-R1 probe in saline-(Figure 3D) and PS-(Figure 3E)-treated rat bladders. Note the higher detection of the anti-VEGF-R1 probe in the saline-treated rat bladder, compared to the PS-treated bladder. SA-HRP was used to target the anti-decorin probe in saline-(Figure 4D) and PS-(Figure 4E)-treated rat bladders. Note the higher detection of the anti-decorin probe in the saline-treated rat bladder, compared to the PS-treated bladder. SA-HRP was used to target the biotin moiety of each probe in ex vivo tissues, which supported the in vivo data.

Figure 1.

Figure 1

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Illustration of the Gd-DTPA-albumin-Ab-biotin probe construct used to generate anti-claudin-2, anti-VEF-R1, and anti-decorin probes.

Figure 2.

Figure 2

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In vivo molecular-targeted MRI of claudin-2 in (A) PS- and (B) sa-line-treated rat bladders (24 h). (ii) Color-enhanced image. (C) Percent change in MRI signal intensities (SI). Significant increase in % MRI SI in PS vs. saline bladders (P<0.05). Streptavidin-HRP targeting biotin moiety of probe in (D) PS- or (E) saline-treated bladders.

Figure 3.

Figure 3

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In vivo molecular-targeted MRI of VEGF-R1 in (A) saline- and (B) PS-treated rat bladders (24 h). (ii) Color-enhanced image. (C) Percent change in MRI signal intensities (SI). Significant increase in % MRI SI in saline vs. PS bladders (P<0.01). Streptavidin-HRP targeting biotin moiety of probe in (D) saline- or (E) PS-treated bladders.

Figure 4.

Figure 4

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In vivo molecular-targeted MRI of decorin in (A) saline- and (B) PS-treated rat bladders (24 h). (ii) Color-enhanced image. (C) Percent change in MRI signal intensities (SI). Significant increase in % MRI SI in saline vs. PS bladders (P<0.05). Streptavidin-HRP targeting biotin moiety of probe in (D) saline- or (E) PS-treated bladders.

Confirmation of IHC levels of each biomarker were also obtained from ex vivo rat bladder sections. Figure 5 depicts levels of claudin-2, VEGF-R1 and decorin in either saline- or PS-treated rat bladders. Note the higher level of claudin-2 staining in the PS-treated rat bladder, compared to the saline control. Note the higher levels of both VEGF-R1 and decorin in the saline control bladders, compared to the PS-treated bladders.

Figure 5.

Figure 5

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Ex vivo immunohistochemistry (IHC) staining for claudin-2, VEGF-R1 and decorin. Saline-treated rat bladders (A: Claudin-2; C: VEGF-R1; and E: Decorin). Protamine sulfate (PS)-treated rat bladders (B: Claudin-2; D: VEGF-R1; and F: Decorin).

Discussion

A Gd-DTPA-albumin-biotin construct (as previously used by our group [38-43]) was used to conjugate an antibody against specific permeability biomarkers to the albumin moiety. In a previous study using a similar Gd-DTPA-albumin-biotin construct with another antibody to detect macromolecular free radicals, we were able to detect ~0.5-1.0 × 10-4 M concentration of the molecularly-targeted probe [38], indicating sensitivity in the micromolar range. From T1 values, estimation of the anti-decorin probe concentration ranges detected were ~0.7-3.0 × 10-4 M, ~0.8-5.0 × 10-4 M for the anti-VEGF-R1 probe, and ~0.2-1.0 × 10-4 M for the anti-claudin-2 probe. The advantage of this construct involves not only assessing in vivo molecular expression assessment of pertinent biomarkers, but also allows direct ex vivo tissue confirmation regarding whether the molecular imaging probe reached its’ intended target. The conjugates were tested in a rat model of permeability in which brief treatment (10 min) with dilute PS (1 mg/ml) is used to induce permeability in the bladder without producing sloughing of the luminal cell layer [34].

Decorin [23,30], VEGF-R1 [31,32], and claudin-2 [33] have been well documented as well-known bladder urothelium markers. Most of these bladder urothelium markers were previously identified by our group (Hurst and co-workers) [19,23,27], and correlated with histology and immunohistochemistry. VEGF-R1 has been previously found as an integral growth factor that stimulates embryonic urinary bladder development [47]. Relevant to IC patients, VEGF-R1 is significantly down-regulated [31]. Decorin, a proteoglycan of chondroitin/dermatan sulfate, has been found to also decrease in feline IC, which is thought to be similar to human IC [23]. On the other hand, claudin-2 has been well documented to increase in cystitis [33,48-50].

Decorin and VEGF-R1 are both expected to decrease (negative response) in PS-induced rat bladders, compared to saline controls, whereas claudin-2 is expected to increase (positive response), compared to saline controls. These observed alterations were supported by the mt-MRI and histological data. MRI- and histologically-detectable levels of decorin and VEGF-R1 were both found to decrease following PS-induced bladder urothelial hyper-permeability. Claudin-2, on the other hand, is over-expressed in relationship to bladder wall hyper-permeability, and was found to increase in the PS model.

Understanding the molecular changes in human patient bladders in IC/BPS has been difficult to achieve because of the necessity of obtaining biopsies for labeling studies. Moreover, evidence from feline bladders suggests that changes in marker distribution may not be uniform over the whole bladder with areas of normal expression coexisting with areas of aberrant expression [23]. This MRI technique should allow more accurate determination of altered biomarker expression in patients that takes spatial distribution into account. Not only can MRI assess the permeability of patient bladders, but also by using molecularly targeted probes, a picture of the molecular changes in defense molecules can be assessed. With these capabilities, better classification of patients and diagnosis should be possible.

In vivo molecularly-targeted MRI (mt-MRI) was found to successfully detect alterations in the expression of decorin, VEGFR1 and claudin-2 in a PS-induced rat bladder permeability model. This in vivo molecular imaging approach can provide invaluable information regarding enhancing our understanding associated with bladder urothelium hyper-permeability in IC patients, and perhaps be used to assist in developing therapeutic strategies.

Acknowledgements

Funding was provided by the Presbyterian Health Foundation (RAT).

Disclosure of conflict of interest

None.

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