SHH regulates penile morphology and smooth muscle through a mechanism involving BMP4 and GREM1.

BACKGROUND: The cavernous nerve (CN) is frequently damaged in prostatectomy and diabetic patients with erectile dysfunction (ED), initiating changes in penile morphology including an acute and intense phase of apoptosis in penile smooth muscle and increased collagen, which alter penile architecture and make corpora cavernosa smooth muscle less able to relax in response to neurotransmitters, resulting in ED. AIM: Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and SHH treatment suppresses penile remodeling after CN injury through an unknown mechanism; we examine if part of the mechanism of how SHH preserves smooth muscle after CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1). METHODS: Primary cultures of smooth muscle cells were established from prostatectomy, diabetic, hypertension and Peyronie's (control) (N = 18) patients. Cultures were characterized by ACTA2, CD31, P4HB, and nNOS immunohistochemical analysis. Patient smooth muscle cell growth was quantified in response to BMP4 and GREM1 treatment. Adult Sprague Dawley rats underwent 1 of 3 surgeries: (1) uninjured or CN-injured rats were treated with BMP4, GREM1, or mouse serum albumin (control) proteins via Affi-Gel beads (N = 16) or peptide amphiphile (PA) (N = 26) for 3 and 14 days, and trichrome stain was performed; (2) rats underwent sham (N = 3), CN injury (N = 9), or CN injury and SHH PA treatment for 1, 2, and 4 days (N = 9). OUTCOMES: Western analysis for BMP4 and GREM1 was performed; (3) rats were treated with 5E1 SHH inhibitor (N = 6) or IgG (control; N = 6) for 2 and 4 days, and BMP4 and GREM1 localization was examined. Statistics were performed by analysis of variance with Scheffé's post hoc test. RESULTS: BMP4 increased patient smooth muscle cell growth, and GREM1 decreased growth. In rats, BMP4 treatment via Affi-Gel beads and PA increased smooth muscle at 3 and 14 days of treatment. GREM1 treatment caused increased collagen and smooth muscle at 3 days, which switched to primarily collagen at 14 days. CN injury increased BMP4 and GREM1, while SHH PA altered Western band size, suggesting alternative cleavage and range of BMP4 and GREM1 signaling. SHH inhibition in rats increased BMP4 and GREM1 in fibroblasts. CLINICAL IMPLICATIONS: Understanding how SHH PA preserves and regenerates penile morphology after CN injury will aid development of ED therapies. STRENGTHS AND LIMITATIONS: SHH treatment alters BMP4 and GREM1 localization and range of signaling, which can affect penile morphology. CONCLUSION: Part of the mechanism of how SHH regulates corpora cavernosa smooth muscle involves BMP4 and GREM1.

BMP4 and GREM1 are targets of SHH signaling and downstream regulators of collagen in the penis.

BACKGROUND: Cavernous nerve (CN) injury, caused by prostatectomy and diabetes, initiates a remodeling process (smooth muscle apoptosis and increased collagen) in the corpora cavernosa of the penis of patients and animal models that is an underlying cause of erectile dysfunction (ED), and the Sonic hedgehog (SHH) pathway plays an essential role in the response of the penis to denervation, as collagen increases with SHH inhibition and decreases with SHH treatment. AIM: We examined if part of the mechanism of how SHH prevents penile remodeling and increased collagen with CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1) and examined the relationship between SHH, BMP4, GREM1, and collagen in penis of ED patients and rat models of CN injury, SHH inhibition, and SHH, BMP4, and GREM1 treatment. METHODS: Corpora cavernosa of Peyronie's disease (control), prostatectomy, and diabetic ED patients were obtained (N = 30). Adult Sprague Dawley rats (n = 90) underwent (1) CN crush (1-7 days) or sham surgery; (2) CN injury and BMP4, GREM1, or mouse serum albumin (control) treatment via Affi-Gel beads or peptide amphiphile (PA) for 14 days; (3) 5E1 SHH inhibitor, IgG, or phosphate-buffered saline (control) treatment for 2 to 4 days; or (4) CN crush with mouse serum albumin or SHH for 9 days. OUTCOMES: Immunohistochemical and Western analysis for BMP4 and GREM1, and collagen analysis by hydroxyproline and trichrome stain were performed. RESULTS: BMP4 and GREM1 proteins were identified in corpora cavernosa smooth muscle of prostatectomy, diabetic, and Peyronie's patients, and in rat smooth muscle, sympathetic nerve fibers, perineurium, blood vessels, and urethra. Collagen decreased 25.4% in rats with CN injury and BMP4 treatment (P = .02) and increased 61.3% with CN injury and GREM1 treatment (P = .005). Trichrome stain showed increased collagen in rats treated with GREM1. Western analysis identified increased BMP4 and GREM1 in corpora cavernosa of prostatectomy and diabetic patients, and after CN injury (1-2 days) in our rat model. Localization of BMP4 and GREM1 changed with SHH inhibition. SHH treatment increased the monomer form of BMP4 and GREM1, altering their range of signaling. CLINICAL IMPLICATIONS: A better understanding of penile remodeling and how fibrosis occurs with loss of innervation is essential for development of novel ED therapies. STRENGTHS AND LIMITATIONS: The relationship between SHH, BMP4, GREM1, and collagen is complex in the penis. CONCLUSION: BMP4 and GREM1 are downstream targets of SHH that impact collagen and may be useful in collaboration with SHH to prevent penile remodeling and ED.

Sonic Hedgehog Signaling in Primary Culture of Human Corpora Cavernosal Tissue From Prostatectomy, Diabetic, and Peyronie's Patients.

BACKGROUND: Cavernous nerve (CN) injury causes penile remodeling, including smooth muscle apoptosis and increased collagen, which results in erectile dysfunction (ED), and prevention of this remodeling is critical for novel ED therapy development. AIM: We developed 2 peptide amphiphile (PA) hydrogel delivery vehicles for Sonic hedgehog (SHH) protein to the penis and CN, which effectively suppress penile distrophic remodeling (apoptosis and fibrosis), in vivo in a rat CN injury model, and the aim of this study is to determine if SHH PA can be used to regenerate human corpora cavernosal smooth muscle deriving from multiple ED origins. METHODS: Corpora cavernosal tissue was obtained from prostatectomy, diabetic, hypertension, cardiovascular disease and Peyronie's (control) patients (n = 21). Primary cultures (n = 21) were established, and corpora cavernosal cells were treated with SHH protein, MSA (control), 5E1 SHH inhibitor, and PBS (control). Growth was quantified by counting the number of cells at 3-4 days. Statistics were performed by ANOVA with Scheffe's post hoc test. Concentration of SHH protein for maximal growth was optimized, and a more active SHH protein examined. OUTCOMES: Cultures were characterized by immunohistochemical analysis with ACTA2, CD31, nNOS and P4HB, and smooth muscle was quantified in comparison to DAPI. RESULTS: Cultures established were >97% smooth muscle. SHH protein increased growth of smooth muscle cells from prostatectomy, diabetic, and Peyronie's patients in a similar manner (49%-51%), and SHH inhibition decreased growth (20%-33%). There was no difference in growth using 25 ug and 10 ug SHH protein, suggesting a threshold concentration of SHH protein above which smooth muscle growth is enhanced. A more active lipid modified SHH peptide further enhanced growth (15%), indicating a more robust growth response. SHH increased growth in smooth muscle cells from hypertension (37%) and cardiovascular disease (32%) patients. SHH protein increased growth under normal and high glucose conditions, suggesting that high glucose conditions that may be present in under controlled diabetic patients would not detract from SHH regenerative capacity. CLINICAL IMPLICATIONS: SHH PA would be beneficial to enhance smooth muscle regeneration in patients with ED of multiple etiologies. STRENGTHS AND LIMITATIONS: Understanding how human corpora cavernosal tissue responds to SHH treatment is critical for clinical translation of SHH PA to ED patients. CONCLUSION: Corpora cavernosal smooth muscle from all ED patients responded to SHH treatment with increased growth. Stupp, SI. Sonic Hedgehog Signaling in Primary Culture of Human Corpora Cavernosal Tissue From Prostatectomy, Diabetic, and Peyronie's Patients. J Sex Med 2022;19:1228-1242.

Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI).

Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. The bacterial FASII pathway is a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. These compounds display an improved low nanomolar enzymatic activity as well as promising low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). The improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.

Identification of Bacillus anthracis PurE inhibitors with antimicrobial activity.

N(5)-carboxy-amino-imidazole ribonucleotide (N(5)-CAIR) mutase (PurE), a bacterial enzyme in the de novo purine biosynthetic pathway, has been suggested to be a target for antimicrobial agent development. We have optimized a thermal shift method for high-throughput screening of compounds binding to Bacillus anthracis PurE. We used a low ionic strength buffer condition to accentuate the thermal shift stabilization induced by compound binding to Bacillus anthracis PurE. The compounds identified were then subjected to computational docking to the active site to further select compounds likely to be inhibitors. A UV-based enzymatic activity assay was then used to select inhibitory compounds. Minimum inhibitory concentration (MIC) values were subsequently obtained for the inhibitory compounds against Bacillus anthracis (ΔANR strain), Escherichia coli (BW25113 strain, wild-type and ΔTolC), Francisella tularensis, Staphylococcus aureus (both methicillin susceptible and methicillin-resistant strains) and Yersinia pestis. Several compounds exhibited excellent (0.05-0.15µg/mL) MIC values against Bacillus anthracis. A common core structure was identified for the compounds exhibiting low MIC values. The difference in concentrations for inhibition and MIC suggest that another enzyme(s) is also targeted by the compounds that we identified.

Adrenergic modulation of cytokine release in bone marrow progenitor-derived macrophage following polymicrobial sepsis.

Catecholamines may impact on the pathophysiology of sepsis by attenuating proinflammatory cytokine and augmenting antiinflammatory cytokine production by macrophages. We tested this premise in bone marrow monocyte progenitor-derived macrophages. Polymicrobial sepsis was induced in mice through cecal ligation and puncture. ER-MP 12 monocyte progenitors were isolated and differentiated into macrophages in vitro 72 hr later. Lipopolysaccharide (LPS)-stimulated cytokine production was measured with and without epinephrine, IL-10 and anti-IL-10 antibody. Epinephrine significantly increased IL-10 production, but attenuated TNF-alpha release exclusively through beta2 adrenergic receptors, and is independent of IL-10 production. Together, these results suggest that epinephrine can promote a potent antiinflammatory response in sepsis.

Thermal injury and sepsis modulates beta-adrenergic receptors and cAMP responses in monocyte-committed bone marrow cells.

We have previously reported that adrenergic stimulation enhances monocytopoiesis following experimental burn injury and sepsis (BI/S). In the present work we measured beta-adrenergic receptor number and affinity in bone marrow committed monocyte progenitor cells (CD59(+)) following BI/S. We find that BI/S treatment significantly decreased monocyte progenitor cell beta-adrenergic receptors but significantly increased receptor binding affinity and isoproterenol-stimulated cAMP production. CD14 expression in macrophages derived in vitro from CD59(+) cells following BI/S was significantly increased by epinephrine and this change was blocked by beta(2)-adrenergic receptor antagonist. PCR analysis suggests the presence of beta(2)- but not beta(1)-adrenergic receptors. Enhanced adrenergic receptor signaling in CD59(+) bone marrow cells following BI/S may be important in macrophage development.

Indole trimers with antibacterial activity against Gram-positive organisms produced using combinatorial biocatalysis.

The I100V isoform of toluene-4-monooxygenase was used to catalyze the oxidative polymerization of anthranil and various indoles under mildly acidic conditions, favoring the production of trimers. Compounds produced in sufficient yield were purified and tested for their ability to inhibit the growth of B. anthracis, E. faecalis, L. monocytogenes, S. aureus, and in some cases, F. tularensis. 15 of the compounds displayed promising antibacterial activity (MIC < 5 µg/ml) against one or more of the strains tested, with the best MIC values being <0.8 µg/ml. All of these compounds had good selectivity, showing minimal cytotoxicity towards HepG2 cells. The structure was solved for six of the compounds that could be crystallized, revealing that minimally two classes of indole based trimers were produced. One compound class produced was a group of substituted derivatives of the natural product 2,2-bis(3-indolyl) indoxyl. The other group of compounds identified was classified as tryptanthrin-like compounds, all having multi-ring pendant groups attached at position 11 of tryptanthrin. One compound of particular interest, SAB-J85, had a structure that suggests that any compound, with a ring structure that can be activated by an oxygenase, might serve as a substrate for combinatorial biocatalysis.

Discovery of a novel and potent class of F. tularensis enoyl-reductase (FabI) inhibitors by molecular shape and electrostatic matching.

Enoyl-acyl carrier protein (ACP) reductase, FabI, is a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates in a final step of the pathway. The absence of this enzyme in humans makes it an attractive target for the development of new antibacterial agents. FabI is known to be unresponsive to structure-based design efforts due to a high degree of induced fit and a mobile flexible loop encompassing the active site. Here we discuss the development, validation, and careful application of a ligand-based virtual screen used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study, four known classes of FabI inhibitors were used as templates for virtual screens that involved molecular shape and electrostatic matching. The program ROCS was used to search a high-throughput screening library for compounds that matched any of the four molecular shape queries. Matching compounds were further refined using the program EON, which compares and scores compounds by matching electrostatic properties. Using these techniques, 50 compounds were selected, ordered, and tested. The tested compounds possessed novel chemical scaffolds when compared to the input query compounds. Several hits with low micromolar activity were identified and follow-up scaffold-based searches resulted in the identification of a lead series with submicromolar enzyme inhibition, high ligand efficiency, and a novel scaffold. Additionally, one of the most active compounds showed promising whole-cell antibacterial activity against several Gram-positive and Gram-negative species, including the target pathogen. The results of a preliminary structure-activity relationship analysis are presented.

Adrenergic modulation of splenic macrophage cytokine release in polymicrobial sepsis.

Enhanced adrenergic stimulation and catecholamine release are important components of the pathophysiology of sepsis. Under physiological conditions, adrenergic stimulation has been shown to be a negative regulator of proinflammatory cytokine production through increasing IL-10 production. Here we have investigated if adrenergic stimulation similarly inhibits TNF-alpha and IL-6 production by splenic macrophages isolated from a polymicrobial sepsis model. Male B(6)D(2)F(1) mice were subjected to sham (S), laparotomy (Lap), and cecal ligation and puncture (CLP) under anesthesia. Splenic macrophages were isolated 72 h after the initial injury and were stimulated with endotoxin (LPS) in the presence and absence of epinephrine. Compared with S and Lap, splenic macrophages from the CLP group produced significantly less TNF-alpha and IL-6 and more IL-10 when stimulated with LPS. Macrophage cultures from CLP animals incubated with either epinephrine or IL-10 for 2 h had significantly reduced TNF-alpha and IL-6 release in response to LPS. However, similar cultures pretreated with IL-10 antibody before the addition of exogenous epinephrine failed to reverse the attenuation of LPS-stimulated cytokines. Pretreatment of macrophage cultures with beta(2)- (ICI-118551) but not beta(1)-adrenergic (atenolol) receptor antagonists reversed the epinephrine-mediated cytokine attenuation following LPS treatment. Data are also presented that demonstrate the involvement of protein kinase A activation with adrenergic agonist but not with IL-10 stimulation. Taken together, these findings suggest that adrenergic mechanisms may influence peripheral tissue macrophage inflammatory cytokine response following trauma and sepsis, independent of the effects of IL-10.

Alpha-melanocyte-stimulating hormone inhibits lung injury after renal ischemia/reperfusion.

Combined acute renal and pulmonary failure has a very high mortality. In animals, lung injury develops after shock or visceral or renal ischemia. Alpha-melanocyte-stimulating hormone (alpha-MSH) is an antiinflammatory cytokine, which inhibits inflammatory, apoptotic, and cytotoxic pathways implicated in acute renal injury. We sought to determine if alpha-MSH inhibits acute lung injury after renal ischemia and to determine the early mechanisms of alpha-MSH action. Mice were subjected to renal ischemia treated with vehicle or alpha-MSH. At early time points, we measured organ histology, leukocyte accumulation, myeloperoxidase activity, activation of nuclear factor-kappaB, p38 mitogen-activated protein kinase, c-Jun, and activator protein-1 pathways, in addition to messenger RNA for intracellular adhesion molecule-1 and tumor necrosis factor-alpha. Renal ischemia rapidly activated kidney and lung nuclear factor-kappaB, p38 mitogen-activated protein kinase, c-Jun, and activator protein-1 pathways, and distant lung injury. Alpha-MSH administration immediately before reperfusion significantly decreased kidney and lung injury and prevented activation of kidney and lung transcription factors and stress response genes, and lung intracellular adhesion molecule-1 and tumor necrosis factor-alpha at early time points after renal ischemia/reperfusion. We conclude that distant lung injury occurs rapidly after renal ischemia. alpha-MSH protects against both kidney and lung damage after renal ischemia, in part, by inhibiting activation of transcription factors and stress genes early after renal injury.

Prostaglandin E2 mediates growth arrest in NFS-60 cells by down-regulating interleukin-6 receptor expression.

Interleukin-6 (IL-6), a potent myeloid mitogen, and the immunosuppressive prostanoid prostaglandin E2 (PGE2) are elevated following thermal injury and sepsis. We have previously demonstrated that bone marrow myeloid commitment shifts toward monocytopoiesis and away from granulocytopoiesis during thermal injury and sepsis and that PGE2 plays a central role in this alteration. Here we investigated whether PGE2 can modulate IL-6-stimulated growth in the promyelocytic cell line, NFS-60, by down-regulating IL-6 receptor (IL-6r) expression. Exposure of NFS-60 cells to PGE2 suppressed IL-6-stimulated proliferation as well as IL-6r expression. Receptor down-regulation is functionally significant since IL-6-induced signal transduction through activators of transcription (STAT)-3 is also decreased. Down-regulation of IL-6r correlated with the ability of PGE2 to arrest cells in the G0/G1 phase of the cell cycle. PGE2 appears to signal through EP2 receptors. Butaprost (EP2 agonist) but not sulprostone (EP3 agonist) inhibited IL-6-stimulated proliferation. In addition, an EP2 antagonist (AH6809) alleviated the anti-proliferative effects of PGE2. NFS-60 cells express predominantly EP2 and EP4 receptors. While PGE2 down-regulated both the IL-6r protein and mRNA expression, it had no influence on EP2 or EP4 mRNA expression. The present study demonstrates that PGE2 is a potent down-regulator of IL-6r expression and thus may provide a mechanistic explanation for the granulocytopenia seen in thermal injury and sepsis.