Francis F.Y. Lam

Functional Mesenchymal Stem Cells Derived From Human Induced Pluripotent Stem Cells Attenuate Limb Ischemia in Mice

Background— Aging and aging-related disorders impair the survival and differentiation potential of bone marrow mesenchymal stem cells (MSCs) and limit their therapeutic efficacy. Induced pluripotent stem cells (iPSCs) may provide an alternative source of functional MSCs for tissue repair. This study aimed to generate and characterize human iPSC-derived MSCs and to investigate their biological function for the treatment of limb ischemia. Methods and Results— Human iPSCs were induced to MSC differentiation with a clinically compliant protocol. Three monoclonal, karyotypically stable, and functional MSC-like cultures were successfully isolated using a combination of CD24− and CD105+ sorting. They did not express pluripotent-associated markers but displayed MSC surface antigens and differentiated into adipocytes, osteocytes, and chondrocytes. Transplanting iPSC-MSCs into mice significantly attenuated severe hind-limb ischemia and promoted vascular and muscle regeneration. The benefits of iPSC-MSCs on limb ischemia were superior to those of adult bone marrow MSCs. The greater potential of iPSC-MSCs may be attributable to their superior survival and engraftment after transplantation to induce vascular and muscle regeneration via direct de novo differentiation and paracrine mechanisms. Conclusions— Functional MSCs can be clonally generated, beginning at a single-cell level, from human iPSCs. Patient-specific iPSC-MSCs can be prepared as an “off-the-shelf” format for the treatment of tissue ischemia.

Pharmacological investigation on the wound healing effects of Radix Rehmanniae in an animal model of diabetic foot ulcer.

ETHNOPHARMACOLOGICAL RELEVANCE Radix Rehmanniae (RR) has a very long history of usage in traditional Chinese medicine and is usually one of the principal herb found in many herbal formulae used in diabetic foot ulcer. AIM OF THE STUDY RR aqueous extract was investigated for its wound healing effects in a diabetic foot ulcer rat model and its detailed mechanism of actions. MATERIALS AND METHODS A previously established diabetic foot ulcer rat model was used to assess the effect of RR extract on wound area reduction, tissue regeneration and angiogenesis. Carrageenan-induced inflammation rat model was used for inflammation study; and diabetic control was evaluated using a neonatal streptozotocin-induced diabetic rat model. RESULTS In the RR treated group, a trend of reduction of the wound area was observed from days 8 to 18 and a significant difference (as compared with control group) was found on day 8. The ulcer healing effect of RR extract was further supported by better developed scars and epithelialization as well as good formation of capillaries with enhanced VEGF expression. Carrageenan-induced inflammation was also significantly alleviated with RR extract. CONCLUSIONS Our results demonstrated for the first time that Radix Rehmanniae was effective in promoting diabetic foot ulcer healing in rats through the processes of tissue regeneration, angiogenesis and inflammation control, but not glycemia control. The present study provided scientific basis to support the traditional use of Radix Rehmanniae in diabetic foot ulcer.

Pharmacological evidence for calcium channel inhibition by danshen (Salvia miltiorrhiza) on rat isolated femoral artery.

This study investigated the relaxant actions of Danshen (Salvia miltiorrhiza) and its lipid-soluble- and water-soluble-fractions on endothelium-denuded rat isolated femoral artery rings. Danshen, its water-soluble fraction and its lipid-soluble fraction produced relaxation of the phenylephrine-precontracted artery rings with IC50 values of 149 ± 20 μg/mL, 160 ± 25 μg/mL, and 23 ± 6 μg/mL, respectively. Pretreatment of the artery rings with a non-selective potassium channel inhibitor tetraethylammonium (TEA, 10 mM) produced a significant two-fold rightward shift of the concentration-response curve to Danshen and a four-fold shift to its water-soluble fraction, but had no effect on the lipid-soluble fraction. A 3.3-fold shift was produced on the concentration-response curve of Danshen when the artery rings were pretreated with a mixture of 10 mM TEA, 1 mM 4-aminopyridine (KV blocker), 1 μM glibenclamide (KATP blocker), 100 nM iberiotoxin (BKCa blocker), and 100 μM barium chloride (KIR blocker). Involvement of Ca2+ channels was investigated in endothelium-denuded artery rings incubated with Ca2+-free buffer and primed with 1 μM phenylephrine or 60 mM KCl for 5 minutes prior to adding CaCl2 to elicit contraction. In artery rings primed with phenylephrine, pretreatment with 1 mg/mL Danshen, 1 mg/mL water-soluble fraction of Danshen, 0.1 mg/mL lipid-soluble fraction of Danshen, and 100 nM nifedipine abrogated the CaCl2-induced contraction. On the other hand, in artery rings primed with KCl, these agents produced 40%, 25%, 53%, and 92% inhibition on the maximum contraction induced by CaCl2, respectively. Increasing the concentrations of Danshen and its water-soluble fraction to 3 mg/mL, and the lipid-soluble fraction to 0.3 mg/mL further reduced the maximum contraction to 92%, 93%, and 83%, respectively. Taken together, these findings suggested the vasorelaxant actions of Danshen and its fractions were produced primarily by inhibition of Ca2+ influx in the vascular smooth muscle cells and a small component was mediated by the opening of K+ channels.

Functional Role of TRPV4-KCa2.3 Signaling in Vascular Endothelial Cells in Normal and Streptozotocin-Induced Diabetic Rats

The small conductance and intermediate conductance Ca2+-activated K+ channels are known to be involved in the endothelium-dependent hyperpolarization. Ca2+ entry into endothelial cells stimulates these channels, causing membrane hyperpolarization in endothelial cells and underlying smooth muscle cells. In the present study, with the use of coimmunoprecipitation and double immunolabeling methods, we demonstrated a physical interaction of transient receptor potential vanilloid 4 (TRPV4) with KCa2.3 in rat mesenteric artery endothelial cells. Acetylcholine and 4&agr;-PDD mainly acted through TRPV4-KCa2.3 pathway to induce smooth muscle hyperpolarization and vascular relaxation. KCa3.1 was also involved in the process but at a much lesser degree than that of KCa2.3. Stimulating TRPV4-KCa2.3 signaling pathway also increased local blood flow in mesenteric beds and reduced systemic blood pressure in anesthetized rats. In streptozotocin-induced diabetic rats, the expression levels of TRPV4 and KCa2.3 were reduced, which could be an underlying reason for the dysfunction of endothelium-dependent hyperpolarization in these animals. These results demonstrated an important physiological and pathological role of TRPV4-KCa2.3 signaling pathway in vascular endothelial cells.

Dihydrotanshinone, a lipophilic component of Salvia miltiorrhiza (danshen), relaxes rat coronary artery by inhibition of calcium channels.

AIM OF THE STUDY Dihydrotanshinone is a lipophilic component of the medicinal herb Salvia miltiorrhiza (danshen) belonging to the family of Labiatae. In this study, we have investigated the mechanisms of its relaxant effect on rat-isolated coronary artery. MATERIALS AND METHODS Rat coronary artery rings were precontracted with 1 microM 5-hydroxytryptamine (5-HT). Involvement of endothelium-dependant mechanisms were investigated by pretreatment of the artery rings with a cyclooxygenase inhibitor flurbiprofen (10 microM), a nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), a muscarinic receptor antagonist atropine (100 nM), and by mechanical removal of the endothelium. Involvement of endothelium-independent mechanisms was investigated in endothelium-denuded artery rings pretreated with a beta-adrenoceptor antagonist propranolol (100 nM), an adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536, 100 microM), a guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ,10 microM), and a potassium channel inhibitor tetraethylammonium (TEA, 10 mM). Involvement of Ca(2+) channels was investigated in artery rings incubated with Ca(2+)-free buffer and primed with 1 microM 5-HT for 5 min prior to adding CaCl(2) to elicit contraction. RESULTS Dihydrotanshinone relaxed the 5-HT-precontracted coronary artery rings with an IC50 value of 10.39+/-1.69 microM. None of the above inhibitors or antagonists tested produced a significant change on the vasorelaxant effect of dihydrotanshinone, except ODQ caused a 50% reduction. Pre-incubation of the artery rings for 10 min with dihydrotanshinone (100 microM) abolished the CaCl(2)-induced vasoconstriction. CONCLUSIONS These findings suggest that inhibition of Ca(2+) influx in the vascular smooth muscle cells is important for the vasorelaxant effect of dihydrotanshinone, and it is independent of pathways involving the endothelium, muscarinic receptors, beta-adrenoceptors, adenylyl cyclase, and guanylyl cyclase.

Mechanisms of the dilator action of Danshen (Salvia miltiorrhiza) on rat isolated femoral artery.

This study investigates the actions of Danshen crude extract (Salvia miltiorrhiza) on rat isolated femoral artery rings precontracted with phenylephrine. Low concentrations of Danshen (10 to 30 μg/mL) enhanced the phenylephrine-precontracted tone by a maximum of 31.20 ± 2.71%. At concentrations 100 μg/mL or above, Danshen relaxed the precontracted tone, with full relaxation obtained at 1 mg/mL. Involvement of endothelium-dependant mechanisms in the dilator effect of Danshen was investigated by pretreatment of the artery rings with a cyclooxygenase inhibitor flurbiprofen (10 μM), a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 μM), a muscarinic receptor antagonist atropine (100 nM), and by mechanical removal of the endothelium; none of these procedures produced a significant change on the Danshen-induced effect. Involvement of endothelium-independent mechanisms was investigated in endothelium-denuded artery rings pretreated with a histamine H2 receptor antagonist cimetidine (10 μM), a β-adrenoceptor antagonist propranolol (100 nM), an adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536, 100 μM), a guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM), and a potassium channel inhibitor tetraethylammonium (TEA, 10 and 100 mM); only TEA was effective in partially inhibiting the Danshen-induced effect. These findings suggest the dilator action of Danshen on rat femoral artery was mediated in part by the opening of TEA-sensitive K+ channels in the smooth muscle cells. Muscarinic receptors, histamine receptors, β-adrenoceptors, endothelium-derived relaxant factors, adenylyl cyclase, and guanylyl cyclase-dependent pathways did not play a role in its vasodilatory effect.

Mechanisms of the relaxant effect of a Danshen and Gegen formulation on rat isolated cerebral basilar artery

AIM OF THE STUDY Danshen (root of Salvia miltiorrhiza) and gegen (root of Pueraria lobata) are two herbs used in traditional Chinese medicine, most commonly for their putative cardioprotective and anti-atherosclerotic effects. In this study, the actions of a danshen and gegen formulation (DG; ratio 7:3) were investigated on rat-isolated cerebral basilar artery. MATERIALS AND METHODS Rat basilar artery rings were precontracted with 100 nM U46619. Involvement of endothelium-dependent mechanisms was investigated by mechanical removal of the endothelium; K(+) channels were investigated by pretreatment of the artery rings with various K(+) channel inhibitors, and Ca(2+) channels were investigated in artery rings incubated with Ca(2+)-free buffer and primed with 100 nM U46619 for 5 min prior to adding CaCl(2) to elicit contraction. RESULTS DG produced concentration-dependent relaxation of the artery rings with an IC(50) of 895±121 μg/ml. Mechanical removal of the endothelium or pretreatment with the BK(Ca) channel inhibitor iberiotoxin (100 nM), the K(V) channel inhibitor 4-aminopyridine (1 mM), or the K(IR) channel inhibitor barium chloride (100 μM), all had no effect on the DG-induced response (P>0.05 for all). However, pretreatment with the K(ATP) channel inhibitor glibenclamide (1 μM), the non-selective K(+) channel inhibitor tetraethylammonium (TEA, 100 mM), or a combination of all the K(+) channel inhibitors (iberiotoxin+4-aminopyrindine+barium chloride+glibenclamide+TEA) produced significant inhibition on the DG-induced response (P<0.01 for all); its maximum vasorelaxant effect (Imax) was reduced by 37, 24, and 30%, respectively. Preincubation of the artery rings with DG for 10 min produced concentration-dependent (1, 3 and 7 mg/ml) and total inhibition on the CaCl(2)-induced vasoconstriction. CONCLUSIONS These findings suggest the vasorelaxant effect of DG on rat basilar artery is independent of endothelium-derived mediators, whereas, inhibition of Ca(2+) influx in the vascular smooth muscle cells is important, and a minor component is mediated by the opening of K(ATP) channels. DG could be a useful cerebroprotective agent in some patients with occlusive cerebrovascular disease.

Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms.

The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.

Analgesic and anti-arthritic effects of Lingzhi and San Miao San supplementation in a rat model of arthritis induced by Freund's complete adjuvant.

AIM OF STUDY In this study, we have investigated the analgesic and anti-arthritic effects of a traditional Chinese medicine (TCM) combination of Lingzhi and San Miao San (SMS) in a rat model of arthritis induced by Freunds complete adjuvant (FCA). MATERIALS AND METHODS Sprague-Dawley rats were induced with monoarthritis by single unilateral injection of FCA into the knee joint. The TCM combination was administered to the rats daily by intraperitoneal injection (50mg/(kgday)) or via oral administration (500mg/(kgday)) for 7 days before induction of arthritis and 7 days after. Extension angle that provoked struggling behavior, and size and blood flow of the rat knees were measured to give indexes of allodynia, edema, and hyperemia, respectively. The extent of cell infiltration, tissue proliferation, and erosions of joint cartilage provided additional indexes of the arthritis condition. RESULTS FCA injection produced significant allodynia, edema, hyperemia, immune cell infiltration, synovial tissue proliferation, and erosions of joint cartilage in the ipsilateral knees compared with the contralateral saline-injected knees. Intraperitoneal injection of the TCM combination (50mg/(kgday)) suppressed allodynia, edema, and hyperemia in the inflamed knees, and oral administration (500mg/(kgday)) suppressed edema and hyperemia. Histological examination showed that the TCM administered by either route reduced immune cell infiltration and erosion of joint cartilage. CONCLUSIONS These findings suggest the Lingzhi and SMS formulation has analgesic and anti-inflammatory effects in arthritic rat knees, and concur to previous clinical studies that showed the TCM combination reduced pain in rheumatoid arthritis patients, and extends its possible benefit to suppression of inflammatory symptoms in these patients.

Mechanisms of the cerebral vasodilator actions of isoflavonoids of Gegen on rat isolated basilar artery.

ETHNOPHARMACOLOGICAL RELEVANCE Gegen (root of Pueraria lobata) is used in traditional Chinese medicine for treatment of cardiovascular diseases. In this study, the relaxant actions of three of its isoflavonoids; puerarin, daidzein, and daidzin, were investigated on rat-isolated cerebral basilar artery. MATERIALS AND METHODS Rat basilar artery rings were precontracted with 100 nM U46619. Involvement of endothelium-dependent mechanisms was investigated by mechanical removal of the endothelium and inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX) enzymes. Adenylyl cyclase- and guanylyl cyclase-dependent pathways were investigated using their respective inhibitors 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536) and 1H-[1,2,4]oxadiazolo [4,3-[alpha]]-quinoxalin-1-one (ODQ). K(+) channels were investigated by pretreatment of the artery rings with various K(+) channel inhibitors, and Ca(2+) channels were investigated in artery rings incubated with Ca(2+)-free buffer and primed with 100 nM U46619 for 5 min prior to adding CaCl(2) to elicit contraction. RESULTS Puerarin, daidzein, and daidzin produced concentration-dependent relaxation of the artery rings with concentration that produced 50% inhibition (IC(50)) of 304 ± 49 μM, 20 ± 7 μM, and 140 ± 21 μM, respectively. Removal of the endothelium produced no change on their vasorelaxant responses except the maximum response (I(max)) to puerarin was inhibited by 28%. The NOS inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 100 μM) also produced 45% inhibition on the puerarin-induced vasorelaxant response, but not the COX inhibitor flurbiprofen (10 μM). SQ22536 (100 μM) and ODQ (100μM) did not affect the vasodilator responses to puerarin, daidzein and daidzin, but glibenclamide (1μM), tetraethylammonium (TEA, 100mM) or a combination of K(+) channel inhibitors (100nM iberiotoxin+1mM 4-aminopyridine+100 μM barium chloride+1 μM glibenclamide+100mM TEA) reduced their I(max). The contractile response to CaCl(2) was attenuated by 61% and 34% in the presence of daidzein and daidzin, respectively, whereas, puerarin did not significantly affect the contraction. CONCLUSIONS The vasorelaxant action of daidzein and daidzin involved opening of K(+) channels and inhibition of Ca(2+) influx in the vascular smooth muscle cells. There is no evidence supporting involvement of endothelium-derived relaxing factors (EDRFs) in their actions. In contrast, puerarin produced vasodilatation via an endothelium-dependent mechanism involving nitric oxide production and an endothelium-independent pathway mediated by the opening of K(+) channels. The cerebral vasodilator activities of all these three isoflavonoids may be beneficial to patients with obstructive cerebrovascular diseases.